Compositions and methods for detecting and treating neurological conditions

ABSTRACT

The present invention relates to the NIPA-1 proteins and nucleic acids encoding the NIPA-1 proteins. The present invention further provides assays for the detection of NIPA-1 polymorphisms and mutations associated with disease states, as well as methods of screening for ligands and modulators of NIPA-1 proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/962,587, filed Aug. 8, 2013, issued Mar. 15, 2016 as U.S. Pat. No.9,285,375, which is a continuation of U.S. patent application Ser. No.12/027,834, filed Feb. 7, 2008, issued Aug. 27, 2013 as U.S. Pat. No.8,528,638, which is a continuation of U.S. patent application Ser. No.10/921,742, issued Feb. 19, 2008 as U.S. Pat. No. 7,332,282, whichclaims priority to U.S. Provisional Application No. 60/496,317, filedAug. 19, 2003, each of which are herein incorporated by reference intheir entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NS033645, NS038713and ES010631 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the NIPA-1 proteins and nucleic acidsencoding the NIPA-1 proteins. The present invention further providesassays for the detection of NIPA-1 polymorphisms and mutationsassociated with disease states, as well as methods of screening fortherapeutic agents, ligands, and modulators of NIPA-1 proteins.

BACKGROUND OF THE INVENTION

Hereditary spastic paraplegia (HSP) (also known as Familial SpasticParaparesis and Strumpell-Lorrain syndrome) is not a single diseaseentity but rather a group of clinically and genetically diversedisorders that share the primary feature of progressive, generallysevere, lower extremity spasticity. HSP is classified as “uncomplicated”(symptoms confined to lower extremity weakness, bladder disturbance, andto a lesser extent impaired position sense in the legs); and“complicated” when additional neurologic deficits are present.

Following normal gestation, delivery, and early childhood development,subjects with uncomplicated autosomal dominant HSP develop leg stiffnessand gait disturbance (e.g., stumbling and tripping) due to difficultydorsiflexing the foot and weakness of hip flexion. Although the majorityof patients experience symptom onset in the second through fourthdecades, there is a wide range of age of symptom onset (from infancythrough age 85) (Cooley et al, Clin Gen 38:57-68 (1990); Dun et al,Neurology 44:1274-7 (1994); Hazan et al, Nat Genet 5:163-7 (1993)). Gaitdisturbance progresses insidiously without exacerbations, remissions, orstep-wise worsening. Paresthesiae below the knees are not uncommon.Urinary urgency progressing to urinary incontinence is a frequent,although variable, late manifestation.

Neurologic examination of subjects with uncomplicated HSP reveals normalfacial and extraocular movements and normal fundi. Although jaw jerk maybe brisk in older subjects, there is no speech disturbance, difficultyswallowing or evidence of frank corticobulbar tract dysfunction. Upperextremity muscle tone and strength are normal. In the lower extremities,muscle tone is increased at the hamstrings, quadriceps and ankles.Weakness is most notable at the iliopsoas, tibialis anterior, and to alesser extent, hamstring muscles. Muscle wasting may occur inuncomplicated HSP (Harding A E, J Neurol Neurosurg Psychiatry 44:871-83(1981); Silver J R, J Neurol Neurosurg Psychiatry 29:135-44 (1996);Cross et al, Arch Neurol 16:473-85 (1967); Refsum and Skillicorn,Neurology 4:40-7 (1954)). Peripheral nerves are normal in uncomplicatedHSP although decreased perception of sharp stimuli below the knees isnoted occasionally. Vibratory sense is often diminished mildly in thedistal lower extremities. When present, this is a useful diagnostic signthat helps distinguish HSP from other disorders. Slight terminaldysmetria is observed occasionally on finger-to-nose testing in olderaffected subjects. Deep tendon reflexes may be brisk in the upperextremities but are pathologically increased in the lower extremities.Gait demonstrates circumduction owing to difficulty with hip flexion andankle dorsiflexion. Crossed adductor reflexes, ankle clonus, andextensor plantar responses are present uniformly. Hoffman's andTromner's signs may be observed. High arched feet (pes cavus) aregenerally present and usually prominent in older affected subjects.

The age of symptom onset, rate of symptom progression, and extent ofdisability are variable both within and between HSP kindreds (Durr etal, Neurology 44:1274-7 (1994); Schady and Scheard, Brain 113:709-20(1990); Polo et al, J Neurol Neurosurg Psychiatry 56:175-81 (1993);Holmes and Shaywitz, J Neurol Neurosurg Psychiatry 40:1003-8 (1977)). Incontrast to variable age of symptom onset and extent of disability, thedistribution of neurologic deficits in uncomplicated HSP is invariantand consist of spastic weakness in the legs; variable impairment ofvibratory sense in the feet; and variable urinary bladder disturbance.Additional deficits such as visual disturbance, marked muscle wasting,fasciculations, dementia, seizures, or peripheral neuropathy in subjectsfrom uncomplicated HSP kindreds should not be attributed to variantpresentations of uncomplicated HSP. Rather, such subjects should beevaluated thoroughly for concurrent or alternative neurologic disorders.Some autosomal dominant uncomplicated HSP kindreds that exhibit onset ofprogressive spastic paraplegia in childhood (before age 6 years) andrelatively little progression of symptoms beyond adolescence. Thesepatients often do not experience urinary bladder disturbances andgenerally remain ambulatory (with assistance).

Electrophysiologic studies are useful for assessing peripheral nerve,muscle, dorsal column, and corticospinal tract involvement in HSP(Harding A E, Semin Neurol 13:333-6 (1993)). These studies areparticularly useful for characterizing the extent of involvement sinceautopsies are obtained infrequently. Although results of these studiesare variable, a number of generalizations can be made. Most studiesfound nerve conduction studies to be normal (in contrast to Friedrich'sataxia and some other spinocerebellar ataxias) (Rosenberg R N, ArchNeurol 50:1123-8 (1993)). One study however, showed that subclinicalsensory impairment was common in HSP, with involvement of peripheralnerves, spinal pathways, or both (Schady and Scheard, Brain 113:709-20(1990)). Lower extremity somatosensory evoked potentials (SSEP) showconduction delay in dorsal column fibers (Pelosi et al, J NeurolNeurosurg Psychiatry 54:1099-102 (1991)). Cortical evoked potentialsused to measure neurotransmission in corticospinal tracts show greatlyreduced corticospinal tract conduction velocity and amplitude of evokedpotential (Claus et al, Ann Neurol 28:43-9 (1990); Polo et al, J NeurolNeurosurg Psychiatry 56:175-81 (1993); Schady et al, J Neurol NeurosurgPsychiatry 54:775-9 (1991); Pelosi et al, J Neurol Neurosurg Psychiatry54:1099-102 (1991)). Often, there is no cortical evoked potentialelicited in muscles innervated by lumbar spinal segments, but corticalevoked potentials of the arms are normal or show only mildly reducedconduction velocity. These findings indicate that there are decreasednumbers of corticospinal tract axons reaching the lumbar spinal cord andthat the remaining axons have reduced conduction velocity. Central motorconduction velocity in the upper extremities was normal except for all 5(affected) members of one HSP kindred for whom responses wereconsiderably delayed. Measurement of central motor conduction velocitymay be a useful way of identifying clinical subgroups of HSP.

Currently, there is no specific treatment to prevent, retard, or reverseHSP's progressive disability. Treatments aimed at reducing andpreventing HSP symptoms are needed. In addition, the molecularpathogenesis of HSP is poorly understood. As such, an understanding ofthe molecular pathogenesis surrounding HSP and similar disorders is alsoneeded

SUMMARY OF THE INVENTION

The present invention relates to the NIPA-1 proteins and nucleic acidsencoding the NIPA-1 proteins. The present invention further providesassays for the detection of NIPA-1 polymorphisms and mutationsassociated with disease states, as well as methods of screening fortherapeutic agents, ligands and modulators of NIPA-1 proteins.

Accordingly, in some embodiments, the present invention provides acomposition comprising an isolated and purified nucleic acid sequenceencoding a protein selected from the group consisting of SEQ ID NOs: 3and 4. In some embodiments, the sequence is operably linked to aheterologous promoter. In other embodiments, the sequence is containedwithin a vector. In further embodiments, the vector is within a hostcell.

In still other embodiments, the nucleic acid is selected from the groupconsisting of SEQ ID NO: 1 and variants thereof that are at least 80%identical to SEQ ID NO: 2. In some embodiments, the nucleic acidsequence is selected from the group consisting of SEQ ID NOs: 1 and 2.

The present invention also provides a composition comprising apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 3 and 4 and variants thereof that are at least80% identical to SEQ ID NOs: 3 and 4. In some embodiments, thepolypeptide is at least 90% identical to SEQ ID NOs: 3 and 4. In otherembodiments, the polypeptide is at least 95% identical to SEQ ID NOs: 3and 4. In still other embodiments, the polypeptide is selected from thegroup consisting of SEQ ID NOs: 3 and 4.

The present invention also provides a method of reducing NIPA-1 activitycomprising providing a target cell expressing NIPA-1 protein, and anagent that inhibits NIPA-1, and contacting the target cell with thecomposition thereby reducing NIPA-1 activity. In some embodiments, thecontacting is conducted in vitro. In some embodiments, the agentcomprises a composition comprising a small interfering RNA duplex(siRNA), or a vector encoding said siRNA, that targets the NIPA-1 mRNA.In other embodiments, the target cell is a neurological cell. In furtherembodiments, the contacting is conducted under conditions such that thevector expresses the siRNA in the target cell. In still otherembodiments, the composition further comprises a nucleic acidtransfecting agent.

The present invention also provides a method comprising providing asubject with symptoms of hereditary spastic paraplegia, and an agentthat reduces symptoms of hereditary spastic paraplegia, andadministering the agent to the subject under conditions such that one ormore symptoms of the hereditary spastic paraplegia are reduced. Inpreferred embodiments, the agent comprises a composition comprisingsmall interfering RNA duplexes (siRNAs), or vectors encoding saidsiRNAs, configured to inhibit expression of NIPA-1 protein. In furtherembodiments, the hereditary spastic paraplegia is autosomal dominanthereditary spastic paraplegia. In other embodiments, the agent isadministered intravenous, topically, and orally. In still furtherembodiments, the composition further comprises a nucleic acidtransfecting agent. In still further embodiments, the compositionfurther comprises reagents suitable for topical administration.

The present invention also provides a method comprising providing asubject at risk for hereditary spastic paraplegia, and an agent thatreduces symptoms of hereditary spastic paraplegia, and administering theagent to the subject under conditions such that one or more symptoms ofthe hereditary spastic paraplegia are prevented. In preferredembodiments, the agent comprises a composition comprising smallinterfering RNA duplexes (siRNAs), or a vector encoding said siRNA,configured to inhibit expression of NIPA-1 protein. In some embodiments,the hereditary spastic paraplegia is autosomal dominant hereditaryspastic paraplegia. In other embodiments, the agent is administeredintravenous, topically, and orally. In still further embodiments, thecomposition further comprises a nucleic acid transfecting agent. Instill further embodiments, the composition further comprises reagentssuitable for topical administration.

The present invention further provides a composition comprising acomposition comprising small interfering RNA duplexes (siRNAs), orvectors encoding said siRNA, configured to inhibit expression of NIPA-1protein, and a nucleic acid transfecting agent.

The present invention also provides a kit comprising a composition,wherein said composition inhibits expression of NIPA-1 protein, andprinted material with instructions for employing said composition fortreating a target cell expressing NIPA-1 protein via expression ofNIPA-1 mRNA under conditions such that the NIPA-1 mRNA is cleaved. Infurther embodiments, the composition comprises small interfering RNAduplexes (siRNAs), or vector encoding said siRNAs, configured to inhibitexpression of NIPA-1 protein.

The present invention also provides a method for producing variants ofNIPA-1 comprising providing a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1 and 2, mutagenizing the nucleic acidsequence, and screening the variant for NIPA-1 activity.

The present invention further provides a method for screening compoundsfor the ability to alter NIPA-1 activity comprising providing apolypeptide sequence comprising at least a portion of NIPA-1, one ormore test compounds, and combining in any order, the polypeptidesequence comprising at least a portion of NIPA-1, and the one or moretest compounds under conditions such that the polypeptide sequence, andthe test compound interact, and measuring NIPA-1 activity.

The present invention further provides a method for identifyingpharmaceutical agents useful for treating hereditary spasticparaplegias, comprising providing target cells, wherein the target cellscomprise NIPA-1 polypeptide, and a candidate pharmaceutical agent, andexposing the target cells to the candidate pharmaceutical agents,measuring the activity of said NIPA-1 polypeptide of said target cells,and selecting candidate pharmaceutical agents that inhibit the activityof the NIPA-1 polypeptide. In other embodiments, the method is used foridentifying hereditary spastic paraplegias, and other motor neurondiseases including, but not limited to, amyotrophic lateral sclerosisand primary lateral sclerosis, and other neurologic disorders,including, but not limited to, spinal chord injury, peripheral nervedisorders, and cerebral palsy.

The present invention also provides a method for diagnosing hereditaryspastic paraplegia, comprising detecting the presence or absence of apolymorphism associated with NIPA-1 gene in a sample. In someembodiments, the polymorphism is in the coding region of said NIPA-1gene. In further embodiments, the polymorphism is a C to G change atposition 159 of SEQ ID NO:5. In still further embodiments, thepolymorphism is in linkage disequilibrium with a C to G change atposition 159 of SEQ ID NO: 5. In other embodiments, the polymorphicprotein comprises additional NIPA-1 amino acid changes.

In other embodiments, the polymorphism disturbs NIPA-1 mRNA compositionor stability. In other preferred embodiments, the polymorphism altersNIPA-1 protein sequence including amino acid substitutions, prematureprotein termination, and aberrant NIPA-1 mRNA splicing leading toaltered NIPA-1 protein sequence.

In other embodiments, the detecting comprises detecting the polymorphismin a nucleic acid from said sample. In further embodiments, the sampleis DNA. In other embodiments, the sample is RNA.

In further embodiments, the detecting comprises detecting a polymorphicprotein. In still further embodiments, the detecting a polymorphicprotein occurs with an antibody. In yet other embodiments, thepolymorphic protein comprises amino acid change threonine to arginine atposition 45 of SEQ ID NO:3 (or position 53 of SEQ ID NO: 7).

The present invention also provides a method for diagnosing hereditaryspastic paraplegia, comprising detecting the presence or absence of aNIPA-1 gene sequence variation in a sample. In some embodiments, theNIPA-1 gene sequence variation is in the coding region of said NIPA-1gene. In further embodiments, the NIPA-1 gene sequence variation is a Cto G change at position 159. In still further embodiments, the NIPA-1gene sequence variation is in linkage disequilibrium with a C to Gchange at position 159.

In other embodiments, the NIPA-1 gene sequence variation disturbs NIPA-1mRNA composition or stability. In other preferred embodiments, theNIPA-1 gene sequence variation alters NIPA-1 protein sequence includingamino acid substitutions, premature protein termination, and aberrantNIPA-1 mRNA splicing leading to altered NIPA-1 protein sequence.

In other embodiments, the detecting comprises detecting the NIPA-1 genesequence variation in a nucleic acid from said sample. In furtherembodiments, the sample is DNA. In other embodiments, the sample is RNA.

In further embodiments, the detecting comprises detecting a polymorphicprotein. In still further embodiments, the detecting a polymorphicprotein occurs with an antibody. In yet other embodiments, thepolymorphic protein comprises amino acid change threonine to arginine atposition 45 of SEQ ID NO:3 (or position 53 of SEQ ID NO: 7). In otherembodiments, the polymorphic protein comprises additional NIPA-I aminoacid changes.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a representative NIPAI exon 1 sequence (SEQ ID NO:6 (ADHSPaffected subject); SEQ ID NO:5 (control subject)).

FIG. 2 shows ADHSP kindreds showing NIPA1 sequence at cDNA position 159(SEQ ID NO:6 (ADHSP affected subject); SEQ ID NO:5 (control subject)).

FIG. 3 shows SPG6 occurs in regions deleted in Prader-Willi (PWS) andAngleman syndromes (AS).

FIG. 4 shows the NIPA1 secondary structure.

FIG. 5 shows expression of NIPA1 by Northern Blot analysis.

FIG. 6 shows the nucleic acid sequence of NIPA-1 (SEQ ID NO: 1)beginning with the start codon.

FIG. 7 shows a variant nucleic acid sequence of NIPA-1 (SEQ ID NO: 2)beginning with the start codon.

FIG. 8 shows the amino acid sequence of NIPA-1 (SEQ ID NO: 3).

FIG. 9 shows a variant amino acid sequence of NIPA-1 (SEQ ID NO: 4).

FIG. 10 shows the nucleic acid sequence of NIPA-1 (SEQ ID NO: 5).

FIG. 11 shows a variant nucleic acid sequence of NIPA-1 (SEQ ID NO: 6).

FIG. 12 shows the amino acid sequence of the wild type NIPA-1 (SEQ IDNO: 7).

FIG. 13 shows a variant amino acid sequence of the mutant NIPA-1 (SEQ IDNO: 8).

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “NIPA-1” when used in reference to a protein ornucleic acid refers to a NIPA-1 protein or nucleic acid encoding aNIPA-1 protein of the present invention. The term NIPA-1 encompassesboth proteins that are identical to wild-type NIPA-1s and those that arederived from wild type NIPA-1 (e.g., variants of NIPA-1 polypeptides ofthe present invention) or chimeric genes constructed with portions ofNIPA-1 coding regions). In some embodiments, the “NIPA-1” is a wild typeNIPA-1 nucleic acid (SEQ ID NO: 1) or amino acid (SEQ ID NO: 3)sequence. In other embodiments, the “NIPA-1” is a variant or mutantnucleic acid (SEQ ID NO: 2) or amino acid (SEQ ID NO: 4).

As used herein, the terms “subject” and “patient” refer to any animal,such as a mammal like a dog, cat, bird, livestock, and preferably ahuman. Specific examples of “subjects” and “patients” include, but arenot limited to, individuals with a hereditary spastic paraplegia (HSP),and individuals with HSP-related characteristics or symptoms.

As used herein, the phrase “symptoms of HSP” and “characteristics ofHSP” include, but are not limited to, lower extremity weakness, bladderdisturbance, impaired position sense in the legs, and neurologicdeficits.

The phrase “under conditions such that the symptoms are reduced” refersto any degree of qualitative or quantitative reduction in detectablesymptoms of HSP, including but not limited to, a detectable impact onthe rate of recovery from disease, or the reduction of at least onesymptom of HSP.

The term “siRNAs” refers to short interfering RNAs. Methods for the useof siRNAs are described in U.S. Patent App. No.: 20030148519/A1 (hereinincorporated by reference). In some embodiments, siRNAs comprise aduplex, or double-stranded region, of about 18-25 nucleotides long;often siRNAs contain from about two to four unpaired nucleotides at the3′ end of each strand. At least one strand of the duplex ordouble-stranded region of a siRNA is substantially homologous to orsubstantially complementary to a target RNA molecule. The strandcomplementary to a target RNA molecule is the “antisense strand;” thestrand homologous to the target RNA molecule is the “sense strand,” andis also complementary to the siRNA antisense strand. siRNAs may alsocontain additional sequences; non-limiting examples of such sequencesinclude linking sequences, or loops, as well as stem and other foldedstructures. siRNAs appear to function as key intermediaries intriggering RNA interference in invertebrates and in vertebrates, and intriggering sequence-specific RNA degradation during posttranscriptionalgene silencing in plants.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression by siRNAs. It is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi may also be considered to inhibit the function of a target RNA; thefunction of the target RNA may be complete or partial.

As used herein, the term “instructions for using said kit for saiddetecting the presence or absence of a variant NIPA-1 nucleic acid orpolypeptide in said biological sample” includes instructions for usingthe reagents contained in the kit for the detection of variant and wildtype NIPA-1 nucleic acids or polypeptides. In some embodiments, theinstructions further comprise the statement of intended use required bythe U.S. Food and Drug Administration (FDA) in labeling in vitrodiagnostic products. The FDA classifies in vitro diagnostics as medicaldevices and requires that they be approved through the 510(k) procedure.Information required in an application under 510(k) includes: 1) The invitro diagnostic product name, including the trade or proprietary name,the common or usual name, and the classification name of the device; 2)The intended use of the product; 3) The establishment registrationnumber, if applicable, of the owner or operator submitting the 510(k)submission; the class in which the in vitro diagnostic product wasplaced under section 513 of the FD&C Act, if known, its appropriatepanel, or, if the owner or operator determines that the device has notbeen classified under such section, a statement of that determinationand the basis for the determination that the in vitro diagnostic productis not so classified; 4) Proposed labels, labeling and advertisementssufficient to describe the in vitro diagnostic product, its intendeduse, and directions for use. Where applicable, photographs orengineering drawings should be supplied; 5) A statement indicating thatthe device is similar to and/or different from other in vitro diagnosticproducts of comparable type in commercial distribution in the U.S.,accompanied by data to support the statement; 6) A 510(k) summary of thesafety and effectiveness data upon which the substantial equivalencedetermination is based; or a statement that the 510(k) safety andeffectiveness information supporting the FDA finding of substantialequivalence will be made available to any person within 30 days of awritten request; 7) A statement that the submitter believes, to the bestof their knowledge, that all data and information submitted in thepremarket notification are truthful and accurate and that no materialfact has been omitted; 8) Any additional information regarding the invitro diagnostic product requested that is necessary for the FDA to makea substantial equivalency determination. Additional information isavailable at the Internet web page of the U.S. FDA.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, RNA (e.g., including but not limited to, mRNA, tRNA andrRNA) or precursor (e.g., NIPA-1). The polypeptide, RNA, or precursorcan be encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction, etc.) ofthe full-length or fragment are retained. The term also encompasses thecoding region of a structural gene and the sequences located adjacent tothe coding region on both the 5′ and 3′ ends for a distance of about 1kb on either end such that the gene corresponds to the length of thefull-length mRNA. The sequences that are located 5′ of the coding regionand which are present on the mRNA are referred to as 5′ untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′untranslated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

In particular, the term “NIPA-1 gene” or “NIPA-1 genes” refers to thefull-length NIPA-1 nucleotide sequence (e.g., contained in SEQ ID NOs: 1and 2). However, it is also intended that the term encompass fragmentsof the NIPA-1 sequences, mutants of the NIPA-1 sequences, as well asother domains within the full-length NIPA-1 nucleotide sequences.Furthermore, the terms “NIPA-1 nucleotide sequence” or “NIPA-1polynucleotide sequence” encompasses DNA sequences, cDNA sequences, RNA(e.g., mRNA) sequences, and associated regulatory sequences.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified,” “mutant,” “polymorphism,” and “variant” refer to a gene orgene product that displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene or gene product. It is noted that naturally-occurringmutants can be isolated; these are identified by the fact that they havealtered characteristics when compared to the wild-type gene or geneproduct.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides or polynucleotidesin a manner such that the 5′ phosphate of one mononucleotide pentosering is attached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotides orpolynucleotide, referred to as the “5′ end” if its 5′ phosphate is notlinked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequentmononucleotide pentose ring. As used herein, a nucleic acid sequence,even if internal to a larger oligonucleotide or polynucleotide, also maybe said to have 5′ and 3′ ends. In either a linear or circular DNAmolecule, discrete elements are referred to as being “upstream” or 5′ ofthe “downstream” or 3′ elements. This terminology reflects the fact thattranscription proceeds in a 5′ to 3′ fashion along the DNA strand. Thepromoter and enhancer elements that direct transcription of a linkedgene are generally located 5′ or upstream of the coding region. However,enhancer elements can exert their effect even when located 3′ of thepromoter element and the coding region. Transcription termination andpolyadenylation signals are located 3′ or downstream of the codingregion.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or, in other words, the nucleic acid sequencethat encodes a gene product. The coding region may be present in a cDNA,genomic DNA, or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements include splicing signals,polyadenylation signals, termination signals, etc.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence5′-“A-G-T-3′,” is complementary to the sequence 3′-“T-C-A-S′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids. Complementarity can include the formationof base pairs between any type of nucleotides, including non-naturalbases, modified bases, synthetic bases and the like.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to inhibition of binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “competes for binding” is used in reference toa first polypeptide with an activity which binds to the same substrateas does a second polypeptide with an activity, where the secondpolypeptide is a variant of the first polypeptide or a related ordissimilar polypeptide. The efficiency (e.g., kinetics orthermodynamics) of binding by the first polypeptide may be the same asor greater than or less than the efficiency substrate binding by thesecond polypeptide. For example, the equilibrium binding constant(K_(D)) for binding to the substrate may be different for the twopolypeptides. The term “K_(m)” as used herein refers to theMichaelis-Menton constant for an enzyme and is defined as theconcentration of the specific substrate at which a given enzyme yieldsone-half its maximum velocity in an enzyme catalyzed reaction.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with about 85-100% identity, preferably about70-100% identity). With medium stringency conditions, nucleic acid basepairing will occur between nucleic acids with an intermediate frequencyof complementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with about 50-70%identity). Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The present invention is not limited to the hybridization of probes ofabout 500 nucleotides in length. The present invention contemplates theuse of probes between approximately 10 nucleotides up to severalthousand (e.g., at least 5000) nucleotides in length. One skilled in therelevant understands that stringency conditions may be altered forprobes of other sizes (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985] and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY[1989]).

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman [Smithand Waterman, Adv. Appl. Math. 2: 482 (1981)] by the homology alignmentalgorithm of Needleman and Wunsch [Needleman and Wunsch, J. Mol. Biol.48:443 (1970)], by the search for similarity method of Pearson andLipman [Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444(1988)], by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length sequences of thecompositions claimed in the present invention (e.g., NIPA-1).

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions that are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion as compared to thenative protein, but where the remaining amino acid sequence is identicalto the corresponding positions in the amino acid sequence deduced from afull-length cDNA sequence. Fragments typically are at least 4 aminoacids long, preferably at least 20 amino acids long, usually at least 50amino acids long or longer, and span the portion of the polypeptiderequired for intermolecular binding of the compositions (claimed in thepresent invention) with its various ligands and/or substrates.

The term “polymorphic locus” is a locus present in a population thatshows variation between members of the population (i.e., the most commonallele has a frequency of less than 0.95). In contrast, a “monomorphiclocus” is a genetic locus at little or no variations seen betweenmembers of the population (generally taken to be a locus at which themost common allele exceeds a frequency of 0.95 in the gene pool of thepopulation).

As used herein, the term “genetic variation information” or “geneticvariant information” refers to the presence or absence of one or morevariant nucleic acid sequences (e.g., polymorphism or mutations) in agiven allele of a particular gene (e.g., a NIPA-1 gene of the presentinvention).

As used herein, the term “detection assay” refers to an assay fordetecting the presence or absence of variant nucleic acid sequences(e.g., polymorphisms or mutations) in a given allele of a particulargene (e.g., a NIPA-1 gene). Examples of suitable detection assaysinclude, but are not limited to, those described below in Section III B.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038[1972]). Other nucleic acid will not be replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (Chamberlin etal., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzymewill not ligate the two oligonucleotides or polynucleotides, where thereis a mismatch between the oligonucleotide or polynucleotide substrateand the template at the ligation junction (D. Y. Wu and R. B. Wallace,Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue oftheir ability to function at high temperature, are found to display highspecificity for the sequences bounded and thus defined by the primers;the high temperature results in thermodynamic conditions that favorprimer hybridization with the target sequences and not hybridizationwith non-target sequences (H. A. Erlich (ed.), PCR Technology, StocktonPress [1989]).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids that may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample that is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template that may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.Particular examples of primers useful in the present invention include,but are not limited to, a primer of least 5 nucleotides from SEQ ID NOs:1 or 2, a primer of at least 10 nucleotides from SEQ ID NOs: 1 or 2, aprimer of at least 20 nucleotides from SEQ ID NOs: 1 or 2, a primer ofat least 30 nucleotides in length from SEQ ID NOs: 1 or 2, a primer ofat least 40 nucleotides in length from SEQ ID NOs: 1 or 2, a primer ofat least 55 nucleotides in length from SEQ ID NOs: 1 or 2, and a primerof at least 50 nucleotides in length from SEQ ID NOs: 1 or 2.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that any probe used in the presentinvention will be labeled with any “reporter molecule,” so that isdetectable in any detection system, including, but not limited to enzyme(e.g., ELISA, as well as enzyme-based histochemical assays),fluorescent, radioactive, and luminescent systems. It is not intendedthat the present invention be limited to any particular detection systemor label.

As used herein, the term “target,” refers to a nucleic acid sequence orstructure to be detected or characterized. Thus, the “target” is soughtto be sorted out from other nucleic acid sequences. A “segment” isdefined as a region of nucleic acid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188, hereby incorporated by reference, that describe a method forincreasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing, and polymerase extension can be repeated many times(i.e., denaturation, annealing and extension constitute one “cycle”;there can be numerous “cycles”) to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template, and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule that is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

As used herein, the term “antisense” is used in reference to RNAsequences that are complementary to a specific RNA sequence (e.g.,mRNA). Included within this definition are antisense RNA (“asRNA”)molecules involved in gene regulation by bacteria. Antisense RNA may beproduced by any method, including synthesis by splicing the gene(s) ofinterest in a reverse orientation to a viral promoter that permits thesynthesis of a coding strand. Once introduced into an embryo, thistranscribed strand combines with natural mRNA produced by the embryo toform duplexes. These duplexes then block either the furthertranscription of the mRNA or its translation. In this manner, mutantphenotypes may be generated. The term “antisense strand” is used inreference to a nucleic acid strand that is complementary to the “sense”strand. The designation (−) (i.e., “negative”) is sometimes used inreference to the antisense strand, with the designation (+) sometimesused in reference to the sense (i.e., “positive”) strand.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding NIPA-1 includes, by way of example, suchnucleic acid in cells ordinarily expressing NIPA-1 where the nucleicacid is in a chromosomal location different from that of natural cells,or is otherwise flanked by a different nucleic acid sequence than thatfound in nature. The isolated nucleic acid, oligonucleotide, orpolynucleotide may be present in single-stranded or double-strandedform. When an isolated nucleic acid, oligonucleotide or polynucleotideis to be utilized to express a protein, the oligonucleotide orpolynucleotide will contain at a minimum the sense or coding strand(i.e., the oligonucleotide or polynucleotide may single-stranded), butmay contain both the sense and anti-sense strands (i.e., theoligonucleotide or polynucleotide may be double-stranded).

As used herein, a “portion of a chromosome” refers to a discrete sectionof the chromosome. Chromosomes are divided into sites or sections bycytogeneticists as follows: the short (relative to the centromere) armof a chromosome is termed the “p” arm; the long arm is termed the “q”arm. Each arm is then divided into 2 regions termed region 1 and region2 (region 1 is closest to the centromere). Each region is furtherdivided into bands. The bands may be further divided into sub-bands. Forexample, the 11p15.5 portion of human chromosome 11 is the portionlocated on chromosome 11 (11) on the short arm (p) in the first region(1) in the 5th band (5) in sub-band 5 (0.5). A portion of a chromosomemay be “altered;” for instance the entire portion may be absent due to adeletion or may be rearranged (e.g., inversions, translocations,expanded or contracted due to changes in repeat regions). In the case ofa deletion, an attempt to hybridize (i.e., specifically bind) a probehomologous to a particular portion of a chromosome could result in anegative result (i.e., the probe could not bind to the sample containinggenetic material suspected of containing the missing portion of thechromosome). Thus, hybridization of a probe homologous to a particularportion of a chromosome may be used to detect alterations in a portionof a chromosome.

The term “sequences associated with a chromosome” means preparations ofchromosomes (e.g., spreads of metaphase chromosomes), nucleic acidextracted from a sample containing chromosomal DNA (e.g., preparationsof genomic DNA); the RNA that is produced by transcription of geneslocated on a chromosome (e.g., hnRNA and mRNA), and cDNA copies of theRNA transcribed from the DNA located on a chromosome. Sequencesassociated with a chromosome may be detected by numerous techniquesincluding probing of Southern and Northern blots and in situhybridization to RNA, DNA, or metaphase chromosomes with probescontaining sequences homologous to the nucleic acids in the above listedpreparations.

As used herein the term “portion” when in reference to a nucleotidesequence (as in “a portion of a given nucleotide sequence”) refers tofragments of that sequence. The fragments may range in size from fournucleotides to the entire nucleotide sequence minus one nucleotide (10nucleotides, 20, 30, 40, 50, 100, 200, etc.).

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets, whichspecify stop codons (i.e., TAA, TAG, TGA).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, NIPA-1 antibodies arepurified by removal of contaminating non-immunoglobulin proteins; theyare also purified by the removal of immunoglobulin that does not bind aNIPA-1 polypeptide. The removal of non-immunoglobulin proteins and/orthe removal of immunoglobulins that do not bind a NIPA-1 polypeptideresults in an increase in the percent of NIPA-1-reactive immunoglobulinsin the sample. In another example, recombinant NIPA-1 polypeptides areexpressed in bacterial host cells and the polypeptides are purified bythe removal of host cell proteins; the percent of recombinant NIPA-1polypeptides is thereby increased in the sample.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant protein” or “recombinant polypeptide” as usedherein refers to a protein molecule that is expressed from a recombinantDNA molecule.

The term “native protein” as used herein, is used to indicate a proteinthat does not contain amino acid residues encoded by vector sequences;that is the native protein contains only those amino acids found in theprotein as it occurs in nature. A native protein may be produced byrecombinant means or may be isolated from a naturally occurring source.

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four consecutive amino acid residues tothe entire amino acid sequence minus one amino acid.

The term “Southern blot,” refers to the analysis of DNA on agarose oracrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Press, NY, pp 9.31-9.58 [1989]).

The term “Northern blot,” as used herein refers to the analysis of RNAby electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (J. Sambrook, et al., supra, pp 7.39-7.52[1989]).

The term “Western blot” refers to the analysis of protein(s) (orpolypeptides) immobilized onto a support such as nitrocellulose or amembrane. The proteins are run on acrylamide gels to separate theproteins, followed by transfer of the protein from the gel to a solidsupport, such as nitrocellulose or a nylon membrane. The immobilizedproteins are then exposed to antibodies with reactivity against anantigen of interest. The binding of the antibodies may be detected byvarious methods, including the use of radiolabeled antibodies.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies that bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

The term “transgene” as used herein refers to a foreign, heterologous,or autologous gene that is placed into an organism by introducing thegene into newly fertilized eggs or early embryos. The term “foreigngene” refers to any nucleic acid (e.g., gene sequence) that isintroduced into the genome of an animal by experimental manipulationsand may include gene sequences found in that animal so long as theintroduced gene does not reside in the same location as does thenaturally-occurring gene. The term “autologous gene” is intended toencompass variants (e.g., polymorphisms or mutants) of the naturallyoccurring gene. The term transgene thus encompasses the replacement ofthe naturally occurring gene with a variant form of the gene.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis (See,Example 10, for a protocol for performing Northern blot analysis).Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the RAD50mRNA-specific signal observed on Northern blots). The amount of mRNApresent in the band corresponding in size to the correctly splicedNIPA-1 transgene RNA is quantified; other minor species of RNA whichhybridize to the transgene probe are not considered in thequantification of the expression of the transgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973]),has been modified by several groups to optimize conditions forparticular types of cells. The art is well aware of these numerousmodifications.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encodingNIPA-1s (e.g., SEQ ID NOs:1 and 2) or fragments thereof may be employedas hybridization probes. In this case, the NIPA-1 encodingpolynucleotide sequences are typically employed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., SDS), and othercomponents (e.g., Denhardt's solution, dry milk, salmon sperm DNA,etc.).

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of containing a human chromosome or sequences associated witha human chromosome may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for Northern blotanalysis), cDNA (in solution or bound to a solid support) and the like.A sample suspected of containing a protein may comprise a cell, aportion of a tissue, an extract containing one or more proteins and thelike.

As used herein, the term “response,” when used in reference to an assay,refers to the generation of a detectable signal (e.g., accumulation ofreporter protein, increase in ion concentration, accumulation of adetectable chemical product).

As used herein, the term “reporter gene” refers to a gene encoding aprotein that may be assayed. Examples of reporter genes include, but arenot limited to, luciferase (See, e.g., deWet et al., Mol. Cell. Biol.7:725 [1987] and U.S. Pat. Nos. 6,074,859; 5,976,796; 5,674,713; and5,618,682; all of which are incorporated herein by reference), greenfluorescent protein (e.g., GenBank Accession Number U43284; a number ofGFP variants are commercially available from CLONTECH Laboratories, PaloAlto, Calif.), chloramphenicol acetyltransferase, β-galactosidase,alkaline phosphatase, and horse radish peroxidase.

As used herein, the terms “computer memory” and “computer memory device”refer to any storage media readable by a computer processor. Examples ofcomputer memory include, but are not limited to, RAM, ROM, computerchips, digital video disc (DVDs), compact discs (CDs), hard disk drives(HDD), and magnetic tape.

As used herein, the term “computer readable medium” refers to any deviceor system for storing and providing information (e.g., data andinstructions) to a computer processor. Examples of computer readablemedia include, but are not limited to, DVDs, CDs, hard disk drives,magnetic tape and servers for streaming media over networks.

As used herein, the terms “processor” and “central processing unit” or“CPU” are used interchangeably and refer to a device that is able toread a program from a computer memory (e.g., ROM or other computermemory) and perform a set of steps according to the program.

As used herein, the term “computer implemented method” refers to amethod utilizing a “CPU” and “computer readable medium.”

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the NIPA-1 proteins and nucleic acidsencoding the NIPA-1 proteins. The present invention further providesassays for the detection of therapeutic agents, and for the detection ofNIPA-1 polymorphisms and mutations associated with disease states.Exemplary embodiments of the present invention are described below.

I. NIPA-1 Polynucleotides

As described above, the present invention provides novel NIPA-1 familygenes. Accordingly, the present invention provides nucleic acidsencoding NIPA-1 genes, homologs, variants (e.g., polymorphisms andmutants), including but not limited to, those described in SEQ ID NOs: 1and 2. Table 1 describes exemplary NIPA-1 genes of the presentinvention. In some embodiments, the present invention providespolynucleotide sequences that are capable of hybridizing to SEQ ID NOs:1 and 2 under conditions of low to high stringency as long as thepolynucleotide sequence capable of hybridizing encodes a protein thatretains a biological activity of the naturally occurring NIPA-1s. Insome embodiments, the protein that retains a biological activity ofnaturally occurring NIPA-1 is 70% homologous to wild-type NIPA-1,preferably 80% homologous to wild-type NIPA-1, more preferably 90%homologous to wild-type NIPA-1, and most preferably 95% homologous towild-type NIPA-1. In preferred embodiments, hybridization conditions arebased on the melting temperature (T_(m)) of the nucleic acid bindingcomplex and confer a defined “stringency” as explained above (See e.g.,Wahl, et al., Meth. Enzymol., 152:399-407 [1987], incorporated herein byreference).

In other embodiments of the present invention, additional alleles ofNIPA-1 genes are provided. In preferred embodiments, alleles result froma polymorphism or mutation (i.e., a change in the nucleic acid sequence)and generally produce altered mRNAs or polypeptides whose structure orfunction may or may not be altered. Any given gene may have none, one ormany allelic forms. Common mutational changes that give rise to allelesare generally ascribed to deletions, additions or substitutions ofnucleic acids. Each of these types of changes may occur alone, or incombination with the others, and at the rate of one or more times in agiven sequence. Examples of the alleles of the present invention includethat encoded by SEQ ID NO: 1 (wild type) and disease alleles thereof(e.g., SEQ ID NO: 2). Additional examples include truncation mutations(e.g., such that the encoded mRNA does not produce a complete protein).

In still other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alteran NIPA-1 coding sequence for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing and/orexpression of the gene product. For example, mutations may be introducedusing techniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, to alter glycosylationpatterns, to change codon preference, etc.).

In some embodiments of the present invention, the polynucleotidesequence of NIPA-1 may be extended utilizing the nucleotide sequence invarious methods known in the art to detect upstream sequences such aspromoters and regulatory elements. For example, it is contemplated thatrestriction-site polymerase chain reaction (PCR) will find use in thepresent invention. This is a direct method that uses universal primersto retrieve unknown sequence adjacent to a known locus (Gobinda et al.,PCR Methods Applic., 2:318-22 [1993]). First, genomic DNA is amplifiedin the presence of a primer to a linker sequence and a primer specificto the known region. The amplified sequences are then subjected to asecond round of PCR with the same linker primer and another specificprimer internal to the first one. Products of each round of PCR aretranscribed with an appropriate RNA polymerase and sequenced usingreverse transcriptase.

In another embodiment, inverse PCR can be used to amplify or extendsequences using divergent primers based on a known region (Triglia etal., Nucleic Acids Res., 16:8186 [1988]). The primers may be designedusing Oligo 4.0 (National Biosciences Inc, Plymouth Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68-72° C. The method uses several restriction enzymesto generate a suitable fragment in the known region of a gene. Thefragment is then circularized by intramolecular ligation and used as aPCR template. In still other embodiments, walking PCR is utilized.Walking PCR is a method for targeted gene walking that permits retrievalof unknown sequence (Parker et al., Nucleic Acids Res., 19:3055-60[1991]). The PROMOTERFINDER kit (Clontech) uses PCR, nested primers andspecial libraries to “walk in” genomic DNA. This process avoids the needto screen libraries and is useful in finding intron/exon junctions.

Preferred libraries for screening for full length cDNAs includemammalian libraries that have been size-selected to include largercDNAs. Also, random primed libraries are preferred, in that they willcontain more sequences that contain the 5′ and upstream gene regions. Arandomly primed library may be particularly useful in case where anoligo d(T) library does not yield full-length cDNA. Genomic mammalianlibraries are useful for obtaining introns and extending 5′ sequence.

In other embodiments of the present invention, variants of the disclosedNIPA-1 sequences are provided. In preferred embodiments, variants resultfrom polymorphisms or mutations (i.e., a change in the nucleic acidsequence) and generally produce altered mRNAs or polypeptides whosestructure or function may or may not be altered. Any given gene may havenone, one, or many variant forms. Common mutational changes that giverise to variants are generally ascribed to deletions, additions orsubstitutions of nucleic acids. Each of these types of changes may occuralone, or in combination with the others, and at the rate of one or moretimes in a given sequence.

It is contemplated that it is possible to modify the structure of apeptide having a function (e.g., NIPA-1 function) for such purposes asaltering the biological activity (e.g., altered NIPA-1 function). Suchmodified peptides are considered functional equivalents of peptideshaving an activity of a NIPA-1 peptide as defined herein. A modifiedpeptide can be produced in which the nucleotide sequence encoding thepolypeptide has been altered, such as by substitution, deletion, oraddition. In particularly preferred embodiments, these modifications donot significantly reduce the biological activity of the modified NIPA-1genes. In other words, construct “X” can be evaluated in order todetermine whether it is a member of the genus of modified or variantNIPA-1's of the present invention as defined functionally, rather thanstructurally. In preferred embodiments, the activity of variant NIPA-1polypeptides is evaluated by methods described herein (e.g., thegeneration of transgenic animals or the use of signaling assays).

Moreover, as described above, variant forms of NIPA-1 genes are alsocontemplated as being equivalent to those peptides and DNA moleculesthat are set forth in more detail herein. For example, it iscontemplated that isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid (i.e., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Accordingly, someembodiments of the present invention provide variants of NIPA-1disclosed herein containing conservative replacements. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidscan be divided into four families: (1) acidic (aspartate, glutamate);(2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan);and (4) uncharged polar (glycine, asparagine, glutamine, cysteine,serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosineare sometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalpolypeptide can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (e.g., LASERGENE software, DNASTAR Inc., Madison,Wis.).

As described in more detail below, variants may be produced by methodssuch as directed evolution or other techniques for producingcombinatorial libraries of variants, described in more detail below. Instill other embodiments of the present invention, the nucleotidesequences of the present invention may be engineered in order to alter aNIPA-1 coding sequence including, but not limited to, alterations thatmodify the cloning, processing, localization, secretion, and/orexpression of the gene product. For example, mutations may be introducedusing techniques that are well known in the art (e.g., site-directedmutagenesis to insert new restriction sites, alter glycosylationpatterns, or change codon preference, etc.).

TABLE 1 NIPA-1 Genes NIPA-1 Gene SEQ ID NO (Nucleic acid) SEQ ID NO(Polypeptide) NIPA-1 5 7 NIPA-1*159 6 8II. NIPA-1 Polypeptides

In other embodiments, the present invention provides NIPA-1polynucleotide sequences that encode NIPA-1 polypeptide sequences (e.g.,the polypeptides of SEQ ID NOs: 3 and 4). Other embodiments of thepresent invention provide fragments, fusion proteins or functionalequivalents of these NIPA-1 proteins. In some embodiments, the presentinvention provides mutants of NIPA-1 polypeptides. In still otherembodiments of the present invention, nucleic acid sequencescorresponding to NIPA-1 variants, homologs, and mutants may be used togenerate recombinant DNA molecules that direct the expression of theNIPA-1 variants, homologs, and mutants in appropriate host cells. Insome embodiments of the present invention, the polypeptide may be anaturally purified product, in other embodiments it may be a product ofchemical synthetic procedures, and in still other embodiments it may beproduced by recombinant techniques using a prokaryotic or eukaryotichost (e.g., by bacterial, yeast, higher plant, insect and mammaliancells in culture). In some embodiments, depending upon the host employedin a recombinant production procedure, the polypeptide of the presentinvention may be glycosylated or may be non-glycosylated. In otherembodiments, the polypeptides of the invention may also include aninitial methionine amino acid residue.

In one embodiment of the present invention, due to the inherentdegeneracy of the genetic code, DNA sequences other than thepolynucleotide sequences of SEQ ID NOs:1 and 2 that encode substantiallythe same or a functionally equivalent amino acid sequence, may be usedto clone and express NIPA-1. In general, such polynucleotide sequenceshybridize to SEQ ID NOs:1 and 2 under conditions of high to mediumstringency as described above. As will be understood by those of skillin the art, it may be advantageous to produce NIPA-1-encoding nucleotidesequences possessing non-naturally occurring codons. Therefore, in somepreferred embodiments, codons preferred by a particular prokaryotic oreukaryotic host (Murray et al., Nucl. Acids Res., 17 [1989]) areselected, for example, to increase the rate of NIPA-1 expression or toproduce recombinant RNA transcripts having desirable properties, such asa longer half-life, than transcripts produced from naturally occurringsequence.

1. Vectors for Production of NIPA-1

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. In some embodiments of the presentinvention, vectors include, but are not limited to, chromosomal,nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40,bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectorsderived from combinations of plasmids and phage DNA, and viral DNA suchas vaccinia, adenovirus, fowl pox virus, and pseudorabies). It iscontemplated that any vector may be used as long as it is replicable andviable in the host.

In particular, some embodiments of the present invention providerecombinant constructs comprising one or more of the sequences asbroadly described above (e.g., SEQ ID NOs: 1 and 2). In some embodimentsof the present invention, the constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In still otherembodiments, the heterologous structural sequence (e.g., SEQ ID NOs: 1and 2) is assembled in appropriate phase with translation initiation andtermination sequences. In preferred embodiments of the presentinvention, the appropriate DNA sequence is inserted into the vectorusing any of a variety of procedures. In general, the DNA sequence isinserted into an appropriate restriction endonuclease site(s) byprocedures known in the art.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. Such vectors include, but are notlimited to, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44, PXT1,pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3)Baculovirus—pPbac and pMbac (Stratagene). Any other plasmid or vectormay be used as long as they are replicable and viable in the host. Insome preferred embodiments of the present invention, mammalianexpression vectors comprise an origin of replication, a suitablepromoter and enhancer, and also any necessary ribosome binding sites,polyadenylation sites, splice donor and acceptor sites, transcriptionaltermination sequences, and 5′ flanking non-transcribed sequences. Inother embodiments, DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in theexpression vector is operatively linked to an appropriate expressioncontrol sequence(s) (promoter) to direct mRNA synthesis. Promotersuseful in the present invention include, but are not limited to, the LTRor SV40 promoter, the E. coli lac or trp, the phage lambda P_(L) andP_(R), T3 and T7 promoters, and the cytomegalovirus (CMV) immediateearly, herpes simplex virus (HSV) thymidine kinase, and mousemetallothionein-I promoters and other promoters known to controlexpression of genes in prokaryotic or eukaryotic cells or their viruses.In other embodiments of the present invention, recombinant expressionvectors include origins of replication and selectable markers permittingtransformation of the host cell (e.g., dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, or tetracycline orampicillin resistance in E. coli).

In some embodiments of the present invention, transcription of the DNAencoding the polypeptides of the present invention by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp that act on a promoter to increase its transcription. Enhancersuseful in the present invention include, but are not limited to, theSV40 enhancer on the late side of the replication origin by 100 to 270,a cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosomebinding site for translation initiation and a transcription terminator.In still other embodiments of the present invention, the vector may alsoinclude appropriate sequences for amplifying expression.

2. Host Cells for Production of NIPA-1 Polypeptides

In a further embodiment, the present invention provides host cellscontaining the above-described constructs. In some embodiments of thepresent invention, the host cell is a higher eukaryotic cell (e.g., amammalian or insect cell). In other embodiments of the presentinvention, the host cell is a lower eukaryotic cell (e.g., a yeastcell). In still other embodiments of the present invention, the hostcell can be a prokaryotic cell (e.g., a bacterial cell). Specificexamples of host cells include, but are not limited to, Escherichiacoli, Salmonella typhimurium, Bacillus subtilis, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus, as wellas Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175 [1981]), C127,3T3, 293, 293T, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (See e.g., Davis et al., Basic Methodsin Molecular Biology, [1986]). Alternatively, in some embodiments of thepresent invention, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., [1989].

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. In other embodiments of thepresent invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

3. Purification of NIPA-1 Polypeptides

The present invention also provides methods for recovering and purifyingNIPA-1 polypeptides from recombinant cell cultures including, but notlimited to, ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. In other embodiments of the present invention,protein-refolding steps can be used as necessary, in completingconfiguration of the mature protein. In still other embodiments of thepresent invention, high performance liquid chromatography (HPLC) can beemployed for final purification steps.

The present invention further provides polynucleotides having a codingsequence of a NIPA-1 gene (e.g., SEQ ID NOs: 1 and 2) fused in frame toa marker sequence that allows for purification of the polypeptide of thepresent invention. A non-limiting example of a marker sequence is ahexahistidine tag which may be supplied by a vector, preferably a pQE-9vector, which provides for purification of the polypeptide fused to themarker in the case of a bacterial host, or, for example, the markersequence may be a hemagglutinin (HA) tag when a mammalian host (e.g.,COS-7 cells) is used. The HA tag corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell, 37:767[1984]).

4. Truncation Mutants of NIPA-1 Polypeptide

In addition, the present invention provides fragments of NIPA-1polypeptides (i.e., truncation mutants). In some embodiments of thepresent invention, when expression of a portion of the NIPA-1 protein isdesired, it may be necessary to add a start codon (ATG) to theoligonucleotide fragment containing the desired sequence to beexpressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al., J. Bacteriol., 169:751 [1987]) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al., Proc. Natl. Acad. Sci. USA 84:2718[1990]). Therefore, removal of an N-terminal methionine, if desired, canbe achieved either in vivo by expressing such recombinant polypeptidesin a host which produces MAP (e.g., E. coli or CM89 or S. cerivisiae),or in vitro by use of purified MAP.

5. Fusion Proteins Containing NIPA-1

The present invention also provides fusion proteins incorporating all orpart of the NIPA-1 polypeptides of the present invention. Accordingly,in some embodiments of the present invention, the coding sequences forthe polypeptide can be incorporated as a part of a fusion gene includinga nucleotide sequence encoding a different polypeptide. It iscontemplated that this type of expression system will find use underconditions where it is desirable to produce an immunogenic fragment of aNIPA-1 protein. In some embodiments of the present invention, the VP6capsid protein of rotavirus is used as an immunologic carrier proteinfor portions of a NIPA-1 polypeptide, either in the monomeric form or inthe form of a viral particle. In other embodiments of the presentinvention, the nucleic acid sequences corresponding to the portion of aNIPA-1 polypeptide against which antibodies are to be raised can beincorporated into a fusion gene construct which includes codingsequences for a late vaccinia virus structural protein to produce a setof recombinant viruses expressing fusion proteins comprising a portionof NIPA-1 as part of the virion. It has been demonstrated with the useof immunogenic fusion proteins utilizing the hepatitis B surface antigenfusion proteins that recombinant hepatitis B virions can be utilized inthis role as well. Similarly, in other embodiments of the presentinvention, chimeric constructs coding for fusion proteins containing aportion of a NIPA-1 polypeptide and the poliovirus capsid protein arecreated to enhance immunogenicity of the set of polypeptide antigens(See e.g., EP Publication No. 025949; and Evans et al., Nature 339:385[1989]; Huang et al., J. Virol., 62:3855 [1988]; and Schlienger et al.,J. Virol., 66:2 [1992]).

In still other embodiments of the present invention, the multipleantigen peptide system for peptide-based immunization can be utilized.In this system, a desired portion of NIPA-1 is obtained directly fromorgano-chemical synthesis of the peptide onto an oligomeric branchinglysine core (see e.g., Posnett et al., J. Biol. Chem., 263:1719 [1988];and Nardelli et al., J. Immunol., 148:914 [1992]). In other embodimentsof the present invention, antigenic determinants of the NIPA-1 proteinscan also be expressed and presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression of proteins, such as a NIPA-1 protein of the presentinvention. Accordingly, in some embodiments of the present invention,NIPA-1 polypeptides can be generated as glutathione-S-transferase (i.e.,GST fusion proteins). It is contemplated that such GST fusion proteinswill enable easy purification of NIPA-1 polypeptides, such as by the useof glutathione-derivatized matrices (See e.g., Ausabel et al. (eds.),Current Protocols in Molecular Biology, John Wiley & Sons, NY [1991]).In another embodiment of the present invention, a fusion gene coding fora purification leader sequence, such as a poly-(His)/enterokinasecleavage site sequence at the N-terminus of the desired portion of aNIPA-1 polypeptide, can allow purification of the expressed NIPA-1fusion protein by affinity chromatography using a Ni²⁺ metal resin. Instill another embodiment of the present invention, the purificationleader sequence can then be subsequently removed by treatment withenterokinase (See e.g., Hochuli et al., J. Chromatogr., 411:177 [1987];and Janknecht et al., Proc. Natl. Acad. Sci. USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment ofthe present invention, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, in other embodiments of the present invention, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed to generate a chimeric genesequence (See e.g., Current Protocols in Molecular Biology, supra).

6. Variants of NIPA-1

Still other embodiments of the present invention provide mutant orvariant forms of NIPA-1 polypeptides (i.e., muteins). It is possible tomodify the structure of a peptide having an activity of a NIPA-1polypeptide of the present invention for such purposes as enhancingtherapeutic or prophylactic efficacy, disabling the protein, orstability (e.g., ex vivo shelf life, and/or resistance to proteolyticdegradation in vivo). Such modified peptides are considered functionalequivalents of peptides having an activity of the subject NIPA-1proteins as defined herein. A modified peptide can be produced in whichthe amino acid sequence has been altered, such as by amino acidsubstitution, deletion, or addition.

Moreover, as described above, variant forms (e.g., mutants orpolymorphic sequences) of the subject NIPA-1 proteins are alsocontemplated as being equivalent to those peptides and DNA moleculesthat are set forth in more detail. For example, as described above, thepresent invention encompasses mutant and variant proteins that containconservative or non-conservative amino acid substitutions.

This invention further contemplates a method of generating sets ofcombinatorial mutants of the present NIPA-1 proteins, as well astruncation mutants, and is especially useful for identifying potentialvariant sequences (i.e., mutants or polymorphic sequences) that areinvolved in neurological disorders (e.g., HSP) or resistance toneurological disorders. The purpose of screening such combinatoriallibraries is to generate, for example, novel NIPA-1 variants that canact as either agonists or antagonists, or alternatively, possess novelactivities all together.

Therefore, in some embodiments of the present invention, NIPA-1 variantsare engineered by the present method to provide altered (e.g., increasedor decreased) biological activity. In other embodiments of the presentinvention, combinatorially-derived variants are generated which have aselective potency relative to a naturally occurring NIPA-1. Suchproteins, when expressed from recombinant DNA constructs, can be used ingene therapy protocols.

Still other embodiments of the present invention provide NIPA-1 variantsthat have intracellular half-lives dramatically different than thecorresponding wild-type protein. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular process that result in destruction of, or otherwiseinactivate NIPA-1 polypeptides. Such variants, and the genes whichencode them, can be utilized to alter the location of NIPA-1 expressionby modulating the half-life of the protein. For instance, a shorthalf-life can give rise to more transient NIPA-1 biological effects and,when part of an inducible expression system, can allow tighter controlof NIPA-1 levels within the cell. As above, such proteins, andparticularly their recombinant nucleic acid constructs, can be used ingene therapy protocols.

In still other embodiments of the present invention, NIPA-1 variants aregenerated by the combinatorial approach to act as antagonists, in thatthey are able to interfere with the ability of the correspondingwild-type protein to regulate cell function.

In some embodiments of the combinatorial mutagenesis approach of thepresent invention, the amino acid sequences for a population of NIPA-1homologs, variants or other related proteins are aligned, preferably topromote the highest homology possible. Such a population of variants caninclude, for example, NIPA-1 homologs from one or more species, orNIPA-1 variants from the same species but which differ due to mutationor polymorphisms. Amino acids that appear at each position of thealigned sequences are selected to create a degenerate set ofcombinatorial sequences.

In a preferred embodiment of the present invention, the combinatorialNIPA-1 library is produced by way of a degenerate library of genesencoding a library of polypeptides which each include at least a portionof potential NIPA-1 protein sequences. For example, a mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential NIPA-1 sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g., for phage display) containing the set ofNIPA-1 sequences therein.

There are many ways by which the library of potential NIPA-1 homologsand variants can be generated from a degenerate oligonucleotidesequence. In some embodiments, chemical synthesis of a degenerate genesequence is carried out in an automatic DNA synthesizer, and thesynthetic genes are ligated into an appropriate gene for expression. Thepurpose of a degenerate set of genes is to provide, in one mixture, allof the sequences encoding the desired set of potential NIPA-1 sequences.The synthesis of degenerate oligonucleotides is well known in the art(See e.g., Narang, Tetrahedron Lett., 39:39 [1983]; Itakura et al.,Recombinant DNA, in Walton (ed.), Proceedings of the 3rd ClevelandSymposium on Macromolecules, Elsevier, Amsterdam, pp 273-289 [1981];Itakura et al., Annu. Rev. Biochem., 53:323 [1984]; Itakura et al.,Science 198:1056 [1984]; Ike et al., Nucl. Acid Res., 11:477 [1983]).Such techniques have been employed in the directed evolution of otherproteins (See e.g., Scott et al., Science 249:386 [1980]; Roberts etal., Proc. Natl. Acad. Sci. USA 89:2429 [1992]; Devlin et al., Science249: 404 [1990]; Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378[1990]; each of which is herein incorporated by reference; as well asU.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815; each of which isincorporated herein by reference).

It is contemplated that the NIPA-1 nucleic acids of the presentinvention (e.g., SEQ ID NOs:1 and 2, and fragments and variants thereof)can be utilized as starting nucleic acids for directed evolution. Thesetechniques can be utilized to develop NIPA-1 variants having desirableproperties such as increased or decreased biological activity.

In some embodiments, artificial evolution is performed by randommutagenesis (e.g., by utilizing error-prone PCR to introduce randommutations into a given coding sequence). This method requires that thefrequency of mutation be finely tuned. As a general rule, beneficialmutations are rare, while deleterious mutations are common. This isbecause the combination of a deleterious mutation and a beneficialmutation often results in an inactive enzyme. The ideal number of basesubstitutions for targeted gene is usually between 1.5 and 5 (Moore andArnold, Nat. Biotech., 14, 458 [1996]; Leung et al., Technique, 1:11[1989]; Eckert and Kunkel, PCR Methods Appl., 1:17-24 [1991]; Caldwelland Joyce, PCR Methods Appl., 2:28 [1992]; and Zhao and Arnold, Nuc.Acids. Res., 25:1307 [1997]). After mutagenesis, the resulting clonesare selected for desirable activity (e.g., screened for NIPA-1activity). Successive rounds of mutagenesis and selection are oftennecessary to develop enzymes with desirable properties. It should benoted that only the useful mutations are carried over to the next roundof mutagenesis.

In other embodiments of the present invention, the polynucleotides ofthe present invention are used in gene shuffling or sexual PCRprocedures (e.g., Smith, Nature, 370:324 [1994]; U.S. Pat. Nos.5,837,458; 5,830,721; 5,811,238; 5,733,731; all of which are hereinincorporated by reference). Gene shuffling involves random fragmentationof several mutant DNAs followed by their reassembly by PCR into fulllength molecules. Examples of various gene shuffling procedures include,but are not limited to, assembly following DNase treatment, thestaggered extension process (STEP), and random priming in vitrorecombination. In the DNase mediated method, DNA segments isolated froma pool of positive mutants are cleaved into random fragments with DNaseIand subjected to multiple rounds of PCR with no added primer. Thelengths of random fragments approach that of the uncleaved segment asthe PCR cycles proceed, resulting in mutations in present in differentclones becoming mixed and accumulating in some of the resultingsequences. Multiple cycles of selection and shuffling have led to thefunctional enhancement of several enzymes (Stemmer, Nature, 370:398[1994]; Stemmer, Proc. Natl. Acad. Sci. USA, 91:10747 [1994]; Crameri etal., Nat. Biotech., 14:315 [1996]; Zhang et al., Proc. Natl. Acad. Sci.USA, 94:4504 [1997]; and Crameri et al., Nat. Biotech., 15:436 [1997]).Variants produced by directed evolution can be screened for NIPA-1activity by the methods described herein.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations, and forscreening cDNA libraries for gene products having a certain property.Such techniques will be generally adaptable for rapid screening of thegene libraries generated by the combinatorial mutagenesis orrecombination of NIPA-1 homologs or variants. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected.

7. Chemical Synthesis of NIPA-1 Polypeptides

In an alternate embodiment of the invention, the coding sequence ofNIPA-1 is synthesized, whole or in part, using chemical methods wellknown in the art (See e.g., Caruthers et al., Nucl. Acids Res. Symp.Ser., 7:215 [1980]; Crea and Horn, Nucl. Acids Res., 9:2331 [1980];Matteucci and Caruthers, Tetrahedron Lett., 21:719 [1980]; and Chow andKempe, Nucl. Acids Res., 9:2807 [1981]). In other embodiments of thepresent invention, the protein itself is produced using chemical methodsto synthesize either an entire NIPA-1 amino acid sequence or a portionthereof. For example, peptides can be synthesized by solid phasetechniques, cleaved from the resin, and purified by preparative highperformance liquid chromatography (See e.g., Creighton, ProteinsStructures And Molecular Principles, W H Freeman and Co, New York N.Y.[1983]). In other embodiments of the present invention, the compositionof the synthetic peptides is confirmed by amino acid analysis orsequencing (See e.g., Creighton, supra).

Direct peptide synthesis can be performed using various solid-phasetechniques (Roberge et al., Science 269:202 [1995]) and automatedsynthesis may be achieved, for example, using ABI 431A PeptideSynthesizer (Perkin Elmer) in accordance with the instructions providedby the manufacturer. Additionally, the amino acid sequence of a NIPA-1polypeptide, or any part thereof, may be altered during direct synthesisand/or combined using chemical methods with other sequences to produce avariant polypeptide.

III. Detection of NIPA-1 Alleles

In some embodiments, the present invention provides methods of detectingthe presence of wild type or variant (e.g., mutant or polymorphic)NIPA-1 nucleic acids or polypeptides. The detection of mutant NIPA-1polypeptides finds use in the diagnosis of disease (e.g., inflammatorydisease).

A. Detection of Variant NIPA-1 Alleles

In some embodiments, the present invention provides alleles of NIPA-1that increase a patient's susceptibility to neurological disorders(e.g., hereditary spastic paraplegias). Any mutation that results in analtered phenotype (e.g., increase in spastic paraplegia disease orresistance to spastic paraplegia disease) is within the scope of thepresent invention.

Accordingly, the present invention provides methods for determiningwhether a patient has an increased susceptibility to a neurologicaldisorders (e.g., ADHSP) by determining, directly or indirectly, whetherthe individual has a variant NIPA-1 allele. In other embodiments, thepresent invention provides methods for providing a prognosis ofincreased risk for spastic paraplegia disease to an individual based onthe presence or absence of one or more variant alleles of NIPA-1.

A number of methods are available for analysis of variant (e.g., mutantor polymorphic) nucleic acid or polypeptide sequences. Assays fordetection variants (e.g., polymorphisms or mutations) via nucleic acidanalysis fall into several categories including, but not limited to,direct sequencing assays, fragment polymorphism assays, hybridizationassays, and computer based data analysis. Protocols and commerciallyavailable kits or services for performing multiple variations of theseassays are available. In some embodiments, assays are performed incombination or in hybrid (e.g., different reagents or technologies fromseveral assays are combined to yield one assay). The following exemplaryassays are useful in the present invention: directs sequencing assays,PCR assays, mutational analysis by dHPLC (e.g., available fromTransgenomic, Omaha, Nebr. or Varian, Palo Alto, Calif.), fragmentlength polymorphism assays (e.g., RFLP or CFLP (See e.g. U.S. Pat. Nos.5,843,654; 5,843,669; 5,719,208; and 5,888,780; each of which is hereinincorporated by reference)), hybridization assays (e.g., directdetection of hybridization, detection of hybridization using DNA chipassays (See e.g., U.S. Pat. Nos. 6,045,996; 5,925,525; 5,858,659;6,017,696; 6,068,818; 6,051,380; 6,001,311; 5,985,551; 5,474,796; PCTPublications WO 99/67641 and WO 00/39587, each of which is hereinincorporated by reference), enzymatic detection of hybridization (Seee.g., U.S. Pat. Nos. 5,846,717, 6,090,543; 6,001,567; 5,985,557;5,994,069; 5,962,233; 5,538,848; 5,952,174 and 5,919,626, each of whichis herein incorporated by reference)), polymorphisms detected directlyor indirectly (e.g., detecting sequences (other polymorphisms) that arein linkage disequilibrium with the polymorphism to be identified; forexample, other sequences in the SPG-6 locus may be used; this method isdescribed in U.S. Pat. No. 5,612,179 (herein incorporated by reference))and mass spectrometry assays.

In addition, assays for the detection of variant NIPA-1 proteins finduse in the present invention (e.g., cell free translation methods, Seee.g., U.S. Pat. No. 6,303,337, herein incorporated by reference) andantibody binding assays. The generation of antibodies that specificallyrecognize mutant versus wild type proteins are discussed below.

B. Kits for Analyzing Risk of Neurological Disorders

The present invention also provides kits for determining whether anindividual contains a wild-type or variant (e.g., mutant or polymorphic)allele or polypeptide of NIPA-1. In some embodiments, the kits areuseful determining whether the subject is at risk of developing aneurological disorder (e.g., HSP). The diagnostic kits are produced in avariety of ways. In some embodiments, the kits contain at least onereagent for specifically detecting a mutant NIPA-1 allele or protein. Inpreferred embodiments, the reagent is a nucleic acid that hybridizes tonucleic acids containing the mutation and that does not bind to nucleicacids that do not contain the mutation. In other embodiments, thereagents are primers for amplifying the region of DNA containing themutation. In still other embodiments, the reagents are antibodies thatpreferentially bind either the wild-type or mutant NIPA-1 proteins.

In some embodiments, the kit contains instructions for determiningwhether the subject is at risk for a neurological disorder (e.g, ADHSP).In preferred embodiments, the instructions specify that risk fordeveloping a spastic paraplegia disease is determined by detecting thepresence or absence of a mutant NIPA-1 allele in the subject, whereinsubjects having an mutant allele are at greater risk for developing aspastic paraplegia disease.

The presence or absence of a disease-associated mutation in a NIPA-1gene can be used to make therapeutic or other medical decisions. Forexample, couples with a family history of spastic paraplegia diseasesmay choose to conceive a child via in vitro fertilization andpre-implantation genetic screening. In this case, fertilized embryos arescreened for mutant (e.g., disease associated) alleles of a NIPA-1 geneand only embryos with wild type alleles are implanted in the uterus.

In other embodiments, in utero screening is performed on a developingfetus (e.g., amniocentesis or chorionic villi screening). In still otherembodiments, genetic screening of newborn babies or very young childrenis performed. The early detection of a NIPA-1 allele known to beassociated with a spastic paraplegia disease allows for earlyintervention (e.g., genetic or pharmaceutical therapies).

In some embodiments, the kits include ancillary reagents such asbuffering agents, nucleic acid stabilizing reagents, protein stabilizingreagents, and signal producing systems (e.g., florescence generatingsystems as Fret systems). The test kit may be packaged in any suitablemanner, typically with the elements in a single container or variouscontainers as necessary along with a sheet of instructions for carryingout the test. In some embodiments, the kits also preferably include apositive control sample.

C. Bioinformatics

In some embodiments, the present invention provides methods ofdetermining an individual's risk of developing a neurological disorder(e.g., HSP) based on the presence of one or more variant alleles of aNIPA-1 gene. In some embodiments, the analysis of variant data isprocessed by a computer using information stored on a computer (e.g., ina database). For example, in some embodiments, the present inventionprovides a bioinformatics research system comprising a plurality ofcomputers running a multi-platform object oriented programming language(See e.g., U.S. Pat. No. 6,125,383; herein incorporated by reference).In some embodiments, one of the computers stores genetics data (e.g.,the risk of contacting a spastic paraplegia disease associated with agiven polymorphism, as well as the sequences). In some embodiments, oneof the computers stores application programs (e.g., for analyzing theresults of detection assays). Results are then delivered to the user(e.g., via one of the computers or via the internet.

For example, in some embodiments, a computer-based analysis program isused to translate the raw data generated by the detection assay (e.g.,the presence, absence, or amount of a given NIPA-1 allele orpolypeptide) into data of predictive value for a clinician. Theclinician can access the predictive data using any suitable means. Thus,in some preferred embodiments, the present invention provides thefurther benefit that the clinician, who is not likely to be trained ingenetics or molecular biology, need not understand the raw data. Thedata is presented directly to the clinician in its most useful form. Theclinician is then able to immediately utilize the information in orderto optimize the care of the subject.

The present invention contemplates any method capable of receiving,processing, and transmitting the information to and from laboratoriesconducting the assays, information providers, medical personal, andsubjects. For example, in some embodiments of the present invention, asample (e.g., a biopsy or a serum or urine sample) is obtained from asubject and submitted to a profiling service (e.g., clinical lab at amedical facility, genomic profiling business, etc.), located in any partof the world (e.g., in a country different than the country where thesubject resides or where the information is ultimately used) to generateraw data. Where the sample comprises a tissue or other biologicalsample, the subject may visit a medical center to have the sampleobtained and sent to the profiling center, or subjects may collect thesample themselves (e.g., a urine sample) and directly send it to aprofiling center. Where the sample comprises previously determinedbiological information, the information may be directly sent to theprofiling service by the subject (e.g., an information card containingthe information may be scanned by a computer and the data transmitted toa computer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., presence of wild type ormutant NIPA-1 genes or polypeptides), specific for the diagnostic orprognostic information desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw data, the prepared format may represent a diagnosis orrisk assessment (e.g., likelihood of developing a spastic paraplegiadisease) for the subject, along with recommendations for particulartreatment options. The data may be displayed to the clinician by anysuitable method. For example, in some embodiments, the profiling servicegenerates a report that can be printed for the clinician (e.g., at thepoint of care) or displayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the association of a given NIPA-1 allele with spasticparaplegia diseases.

IV. Generation of NIPA-1 Antibodies

The present invention provides isolated antibodies or antibody fragments(e.g., FAB fragments). Antibodies can be generated to allow for thedetection of a NIPA-1 proteins (e.g., wild type or mutant) of thepresent invention. The antibodies may be prepared using variousimmunogens. In one embodiment, the immunogen is a human NIPA-1 peptideto generate antibodies that recognize human NIPA-1. Such antibodiesinclude, but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, Fab expression libraries, or recombinant (e.g.,chimeric, humanized, etc.) antibodies, as long as it can recognize theprotein. Antibodies can be produced by using a protein of the presentinvention as the antigen according to a conventional antibody orantiserum preparation process.

Various procedures known in the art may be used for the production ofpolyclonal antibodies directed against a NIPA-1 polypeptide. For theproduction of antibody, various host animals can be immunized byinjection with the peptide corresponding to the NIPA-1 epitope includingbut not limited to rabbits, mice, rats, sheep, goats, etc. In apreferred embodiment, the peptide is conjugated to an immunogeniccarrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyholelimpet hemocyanin (KLH)). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and Corynebacterium parvum).

For preparation of monoclonal antibodies directed toward NIPA-1, it iscontemplated that any technique that provides for the production ofantibody molecules by continuous cell lines in culture will find usewith the present invention (See e.g., Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). These include but are not limited to the hybridomatechnique originally developed by Köhler and Milstein (Köhler andMilstein, Nature 256:495-497 [1975]), as well as the trioma technique,the human B-cell hybridoma technique (See e.g., Kozbor et al., Immunol.Tod., 4:72 [1983]), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 [1985]).

In an additional embodiment of the invention, monoclonal antibodies areproduced in germ-free animals utilizing technology such as thatdescribed in PCT/US90/02545). Furthermore, it is contemplated that humanantibodies will be generated by human hybridomas (Cote et al., Proc.Natl. Acad. Sci. USA 80:2026-2030 [1983]) or by transforming human Bcells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, pp. 77-96 [1985]).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing NIPA-1 specificsingle chain antibodies. An additional embodiment of the inventionutilizes the techniques described for the construction of Fab expressionlibraries (Huse et al., Science 246:1275-1281 [1989]) to allow rapid andeasy identification of monoclonal Fab fragments with the desiredspecificity for a NIPA-1 polypeptide.

In other embodiments, the present invention contemplated recombinantantibodies or fragments thereof to the proteins of the presentinvention. Recombinant antibodies include, but are not limited to,humanized and chimeric antibodies. Methods for generating recombinantantibodies are known in the art (See e.g., U.S. Pat. Nos. 6,180,370 and6,277,969 and “Monoclonal Antibodies” H. Zola, BIOS ScientificPublishers Limited 2000. Springer-Verlay New York, Inc., New York; eachof which is herein incorporated by reference).

It is contemplated that any technique suitable for producing antibodyfragments will find use in generating antibody fragments that containthe idiotype (antigen binding region) of the antibody molecule. Forexample, such fragments include but are not limited to: F(ab′)2 fragmentthat can be produced by pepsin digestion of the antibody molecule; Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, and Fab fragments that can be generated by treatingthe antibody molecule with papain and a reducing agent.

In the production of antibodies, it is contemplated that screening forthe desired antibody will be accomplished by techniques known in the art(e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),“sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitation reactions, immuNIPA-liffusion assays, in situ immunoassays(e.g., using colloidal gold, enzyme or radioisotope labels), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay.)

The foregoing antibodies can be used in methods known in the artrelating to the localization and structure of NIPA-1 (e.g., for Westernblotting), measuring levels thereof in appropriate biological samples,etc. The antibodies can be used to detect a NIPA-1 in a biologicalsample from an individual. The biological sample can be a biologicalfluid, such as, but not limited to, blood, serum, plasma, interstitialfluid, urine, cerebrospinal fluid, and the like, containing cells.

The biological samples can then be tested directly for the presence of ahuman NIPA-1 using an appropriate strategy (e.g., ELISA orradioimmunoassay) and format (e.g., microwells, dipstick (e.g., asdescribed in International Patent Publication WO 93/03367), etc.Alternatively, proteins in the sample can be size separated (e.g., bypolyacrylamide gel electrophoresis (PAGE), in the presence or not ofsodium dodecyl sulfate (SDS), and the presence of NIPA-1 detected byimmunoblotting (Western blotting). Immunoblotting techniques aregenerally more effective with antibodies generated against a peptidecorresponding to an epitope of a protein, and hence, are particularlysuited to the present invention.

Another method uses antibodies as agents to alter signal transduction.Specific antibodies that bind to the binding domains of NIPA-1 or otherproteins involved in intracellular signaling can be used to inhibit theinteraction between the various proteins and their interaction withother ligands. Antibodies that bind to the complex can also be usedtherapeutically to inhibit interactions of the protein complex in thesignal transduction pathways leading to the various physiological andcellular effects of NIPA-1. Such antibodies can also be useddiagnostically to measure abnormal expression of NIPA-1, or the aberrantformation of protein complexes, which may be indicative of a diseasestate.

V. Gene Therapy Using NIPA-1

The present invention also provides methods and compositions suitablefor gene therapy to alter NIPA-1 expression, production, or function. Asdescribed above, the present invention provides human NIPA-1 genes andprovides methods of obtaining NIPA-1 genes from other species. Thus, themethods described below are generally applicable across many species. Insome embodiments, it is contemplated that the gene therapy is performedby providing a subject with a wild-type allele of a NIPA-1 gene (i.e.,an allele that does not contain a NIPA-1 disease allele (e.g., free ofdisease causing polymorphisms or mutations). Subjects in need of suchtherapy are identified by the methods described above. In someembodiments, transient or stable therapeutic nucleic acids are used(e.g., antisense oligonucleotides, siRNAs) to reduce or preventexpression of mutant proteins.

Viral vectors commonly used for in vivo or ex vivo targeting and therapyprocedures are DNA-based vectors and retroviral vectors. Methods forconstructing and using viral vectors are known in the art (See e.g.,Miller and Rosman, BioTech., 7:980-990 [1992]). Preferably, the viralvectors are replication defective, that is, they are unable to replicateautonomously in the target cell. In general, the genome of thereplication defective viral vectors that are used within the scope ofthe present invention lack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution (by other sequences, inparticular by the inserted nucleic acid), partial deletion or additionof one or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (i.e., on the isolated DNA) or insitu, using the techniques of genetic manipulation or by treatment withmutagenic agents.

Preferably, the replication defective virus retains the sequences of itsgenome that are necessary for encapsidating the viral particles. DNAviral vectors include an attenuated or defective DNA viruses, including,but not limited to, herpes simplex virus (HSV), papillomavirus, EpsteinBarr virus (EBV), adenovirus, adeno-associated virus (AAV), and thelike. Defective viruses, that entirely or almost entirely lack viralgenes, are preferred, as defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt etal., Mol. Cell. Neurosci., 2:320-330 [1991]), defective herpes virusvector lacking a glycoprotein L gene (See e.g., Patent Publication RD371005 A), or other defective herpes virus vectors (See e.g., WO94/21807; and WO 92/05263); an attenuated adenovirus vector, such as thevector described by Stratford-Perricaudet et al. (J. Clin. Invest.,90:626-630 [1992]; See also, La Salle et al., Science 259:988-990[1993]); and a defective adeno-associated virus vector (Samulski et al.,J. Virol., 61:3096-3101 [1987]; Samulski et al., J. Virol., 63:3822-3828[1989]; and Lebkowski et al., Mol. Cell. Biol., 8:3988-3996 [1988]).

Preferably, for in vivo administration, an appropriate immunosuppressivetreatment is employed in conjunction with the viral vector (e.g.,adenovirus vector), to avoid immuno-deactivation of the viral vector andtransfected cells. For example, immunosuppressive cytokines, such asinterleukin-12 (IL-12), interferon-gamma (IFN-γ), or anti-CD4 antibody,can be administered to block humoral or cellular immune responses to theviral vectors. In addition, it is advantageous to employ a viral vectorthat is engineered to express a minimal number of antigens.

In a preferred embodiment, the vector is an adenovirus vector.Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to type2 or type 5 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animalorigin (See e.g., WO 94/26914). Those adenoviruses of animal origin thatcan be used within the scope of the present invention includeadenoviruses of canine, bovine, murine (e.g., Mavl, Beard et al.,Virol., 75-81 [1990]), ovine, porcine, avian, and simian (e.g., SAV)origin. Preferably, the adenovirus of animal origin is a canineadenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61strain (ATCC VR-800)).

Preferably, the replication defective adenoviral vectors of theinvention comprise the ITRs, an encapsidation sequence and the nucleicacid of interest. Still more preferably, at least the E1 region of theadenoviral vector is non-functional. The deletion in the E1 regionpreferably extends from nucleotides 455 to 3329 in the sequence of theAd5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3 Afragment). Other regions may also be modified, in particular the E3region (e.g., WO 95/02697), the E2 region (e.g., WO 94/28938), the E4region (e.g., WO 94/28152, WO 94/12649 and WO 95/02697), or in any ofthe late genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in theE1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed inEP 185,573, the contents of which are incorporated herein by reference.In another preferred embodiment, the adenoviral vector has a deletion inthe E1 and E4 regions (Ad 3.0). Examples of E1/E4-deleted adenovirusesare disclosed in WO 95/02697 and WO 96/22378. In still another preferredembodiment, the adenoviral vector has a deletion in the E1 region intowhich the E4 region and the nucleic acid sequence are inserted.

The replication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (See e.g., Levrero et al., Gene 101:195 [1991]; EP 185 573;and Graham, EMBO J., 3:2917 [1984]). In particular, they can be preparedby homologous recombination between an adenovirus and a plasmid thatcarries, inter alia, the DNA sequence of interest. The homologousrecombination is accomplished following co-transfection of theadenovirus and plasmid into an appropriate cell line. The cell line thatis employed should preferably (i) be transformable by the elements to beused, and (ii) contain the sequences that are able to complement thepart of the genome of the replication defective adenovirus, preferablyin integrated form in order to avoid the risks of recombination.Examples of cell lines that may be used are the human embryonic kidneycell line 293 (Graham et al., J. Gen. Virol., 36:59 [1977]), whichcontains the left-hand portion of the genome of an Ad5 adenovirus (12%)integrated into its genome, and cell lines that are able to complementthe E1 and E4 functions, as described in applications WO 94/26914 and WO95/02697. Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques that are well known to one ofordinary skill in the art.

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize that can integrate, in a stable and site-specific manner, into thegenome of the cells that they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The AAV genome has been cloned, sequenced andcharacterized. It encompasses approximately 4700 bases and contains aninverted terminal repeat (ITR) region of approximately 145 bases at eachend, which serves as an origin of replication for the virus. Theremainder of the genome is divided into two essential regions that carrythe encapsidation functions: the left-hand part of the genome, thatcontains the rep gene involved in viral replication and expression ofthe viral genes; and the right-hand part of the genome, that containsthe cap gene encoding the capsid proteins of the virus.

The use of vectors derived from the AAVs for transferring genes in vitroand in vivo has been described (See e.g., WO 91/18088; WO 93/09239; U.S.Pat. No. 4,797,368; U.S. Pat. No. 5,139,941; and EP 488 528, all ofwhich are herein incorporated by reference). These publications describevarious AAV-derived constructs in which the rep and/or cap genes aredeleted and replaced by a gene of interest, and the use of theseconstructs for transferring the gene of interest in vitro (into culturedcells) or in vivo (directly into an organism). The replication defectiverecombinant AAVs according to the invention can be prepared byco-transfecting a plasmid containing the nucleic acid sequence ofinterest flanked by two AAV inverted terminal repeat (ITR) regions, anda plasmid carrying the AAV encapsidation genes (rep and cap genes), intoa cell line that is infected with a human helper virus (for example anadenovirus). The AAV recombinants that are produced are then purified bystandard techniques.

In another embodiment, the gene can be introduced in a retroviral vector(e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764, 4,980,289and 5,124,263; all of which are herein incorporated by reference; Mannet al., Cell 33:153 [1983]; Markowitz et al., J. Virol., 62:1120 [1988];PCT/US95/14575; EP 453242; EP178220; Bernstein et al. Genet. Eng., 7:235[1985]; McCormick, BioTechnol., 3:689 [1985]; WO 95/07358; and Kuo etal., Blood 82:845 [1993]). The retroviruses are integrating viruses thatinfect dividing cells. The retrovirus genome includes two LTRs, anencapsidation sequence and three coding regions (gag, poi and env). Inrecombinant retroviral vectors, the gag, poi and env genes are generallydeleted, in whole or in part, and replaced with a heterologous nucleicacid sequence of interest. These vectors can be constructed fromdifferent types of retrovirus, such as, HIV, MoMuLV (“murine Moloneyleukemia virus” MSV (“murine Moloney sarcoma virus”), HaSV (“Harveysarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcomavirus”) and Friend virus. Defective retroviral vectors are alsodisclosed in WO 95/02697.

In general, in order to construct recombinant retroviruses containing anucleic acid sequence, a plasmid is constructed that contains the LTRs,the encapsidation sequence and the coding sequence. This construct isused to transfect a packaging cell line, which cell line is able tosupply in trans the retroviral functions that are deficient in theplasmid. In general, the packaging cell lines are thus able to expressthe gag, poi and env genes. Such packaging cell lines have beendescribed in the prior art, in particular the cell line PA317 (U.S. Pat.No. 4,861,719, herein incorporated by reference), the PsiCRIP cell line(See, WO90/02806), and the GP+envAm-12 cell line (See, WO89/07150). Inaddition, the recombinant retroviral vectors can contain modificationswithin the LTRs for suppressing transcriptional activity as well asextensive encapsidation sequences that may include a part of the gaggene (Bender et al., J. Virol., 61:1639 [1987]). Recombinant retroviralvectors are purified by standard techniques known to those havingordinary skill in the art.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret. al., Proc. Natl. Acad. Sci. USA 84:7413-7417 [1987]; See also,Mackey, et al., Proc. Natl. Acad. Sci. USA 85:8027-8031 [1988]; Ulmer etal., Science 259:1745-1748 [1993]). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold, Science 337:387-388 [1989]). Particularly useful lipidcompounds and compositions for transfer of nucleic acids are describedin WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, hereinincorporated by reference.

Other molecules are also useful for facilitating transfection of anucleic acid in vivo, such as a cationic oligopeptide (e.g.,WO95/21931), peptides derived from DNA binding proteins (e.g.,WO96/25508), or a cationic polymer (e.g., WO95/21931).

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Methods for formulating and administering naked DNA tomammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and5,589,466, both of which are herein incorporated by reference.

DNA vectors for gene therapy can be introduced into the desired hostcells by methods known in the art, including but not limited totransfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter (See e.g., Wu et al., J. Biol.Chem., 267:963 [1992]; Wu and Wu, J. Biol. Chem., 263:14621 [1988]; andWilliams et al., Proc. Natl. Acad. Sci. USA 88:2726 [1991]).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., Hum. Gene Ther., 3:147 [1992]; and Wu and Wu, J. Biol. Chem.,262:4429 [1987]).

VI. Transgenic Animals Expressing Exogenous NIPA-1 Genes and Homologs,Mutants, and Variants Thereof

The present invention contemplates the generation of transgenic animalscomprising an exogenous NIPA-1 gene or homologs, mutants, or variantsthereof. In preferred embodiments, the transgenic animal displays analtered phenotype as compared to wild-type animals. In some embodiments,the altered phenotype is the overexpression of mRNA for a NIPA-1 gene ascompared to wild-type levels of NIPA-1 expression. In other embodiments,the altered phenotype is the decreased expression of mRNA for anendogenous NIPA-1 gene as compared to wild-type levels of endogenousNIPA-1 expression. In some preferred embodiments, the transgenic animalscomprise mutant alleles of NIPA-1. Methods for analyzing the presence orabsence of such phenotypes include Northern blotting, mRNA protectionassays, and RT-PCR. In other embodiments, the transgenic mice have aknock out mutation of a NIPA-1 gene. In preferred embodiments, thetransgenic animals display an altered susceptibility to neurologicaldisorders (e.g., HSP).

Such animals find use in research applications (e.g., identifyingsignaling pathways that a NIPA-1 protein is involved in), as well asdrug screening applications (e.g., to screen for drugs that prevent ortreat neurological disorders). For example, in some embodiments, testcompounds (e.g., a drug that is suspected of being useful to treat aspastic paraplegia disease) are administered to the transgenic animalsand control animals with a wild type NIPA-1 allele and the effectsevaluated. The effects of the test and control compounds on diseasesymptoms are then assessed.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter, which allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will in general also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. U.S. Pat. No.4,873,191 describes a method for the micro-injection of zygotes; thedisclosure of this patent is incorporated herein in its entirety.

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is utilized to transfect oocytes by injecting the retroviralvector into the perivitelline space of the oocyte (U.S. Pat. No.6,080,912, incorporated herein by reference). In other embodiments, thedeveloping non-human embryo can be cultured in vitro to the blastocyststage. During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Van der Putten,supra; Stewart, et al., EMBO J., 6:383 [1987]). Alternatively, infectioncan be performed at a later stage. Virus or virus-producing cells can beinjected into the blastocoele (Jahner et al., Nature 298:623 [1982]).Most of the founders will be mosaic for the transgene sinceincorporation occurs only in a subset of cells that form the transgenicanimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome that generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germline, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner etal., supra [1982]). Additional means of using retroviruses or retroviralvectors to create transgenic animals known to the art involves themicro-injection of retroviral particles or mitomycin C-treated cellsproducing retrovirus into the perivitelline space of fertilized eggs orearly embryos (PCT International Application WO 90/08832 [1990], andHaskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are utilized to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley etal., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065[1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch, Science 240:1468 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

In still other embodiments, homologous recombination is utilized toknock-out gene function or create deletion mutants (e.g., mutants inwhich a particular domain of a NIPA-1 is deleted). Methods forhomologous recombination are described in U.S. Pat. No. 5,614,396,incorporated herein by reference.

VIII. Drug Screening Using NIPA-1

In some embodiments, the isolated nucleic acid and polypeptides ofNIPA-1 genes of the present invention (e.g., SEQ ID NOS: 1-4) andrelated proteins and nucleic acids are used in drug screeningapplications for compounds that alter (e.g., enhance or inhibit) NIPA-1activity and signaling. The present invention further provides methodsof identifying ligands and signaling pathways of the NIPA-1 proteins ofthe present invention.

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, based upon a hydrophobicity analysis ofNIPA-1 family proteins (see Chai et al, Am J Hum Genet (2003 in press)),it is contemplated that NIPA-1 family proteins function as receptors ortransporters.

In some embodiments, the present invention provides methods of screeningcompounds for the ability to alter NIPA-1 activity mediated by naturalligands (e.g., identified using the methods described above). Suchcompounds find use in the treatment of disease mediated by NIPA-1 familymembers (e.g., HSP).

In some embodiments, the present invention provides methods of screeningcompounds for an ability to interact with mutant NIPA-1 nucleic acid(e.g., SEQ ID NO: 2) and/or mutant NIPA-1 polypeptides (e.g., SEQ ID NO:4), while simultaneously not interacting with wild type NIPA-1 nucleicacid (e.g., SEQ ID NO: 1) and/or wild type NIPA-1 polypeptides (e.g.,SEQ ID NO: 3). Such compounds find use in the treatment of neurologicaldisorders facilitated by the presence of mutant forms of NIPA-1 nucleicacids and/or proteins.

In one screening method, the two-hybrid system is used to screen forcompounds (e.g., proteins) capable of altering NIPA-1 function(s) (e.g.,interaction with a binding partner) in vitro or in vivo. In oneembodiment, a GAL4 binding site, linked to a reporter gene such as lacZ,is contacted in the presence and absence of a candidate compound with aGAL4 binding domain linked to a NIPA-1 fragment and a GAL4transactivation domain II linked to a binding partner fragment.Expression of the reporter gene is monitored and a decrease in theexpression is an indication that the candidate compound inhibits theinteraction of a NIPA-1 with the binding partner. Alternately, theeffect of candidate compounds on the interaction of a NIPA-1 with otherproteins (e.g., proteins known to interact directly or indirectly withthe binding partner) can be tested in a similar manner

In some embodiments, the present invention provides methods ofidentifying NIPA-1 binding partners or ligands that utilizeimmunoprecipitation. In some embodiments, antibodies to NIPA-1 proteinsare utilized to immunoprecipitated NIPA-1s and any bound proteins. Inother embodiments, NIPA-1 fusion proteins are generated with tags andantibodies to the tags are utilized for immunoprecipitation. Potentialbinding partners that immunoprecipitate with NIPA-1s can be identifiedusing any suitable method.

In another screening method, candidate compounds are evaluated for theirability to alter NIPA-1 activity by contacting NIPA-1, binding partners,binding partner-associated proteins, or fragments thereof, with thecandidate compound and determining binding of the candidate compound tothe peptide. The protein or protein fragments is/are immobilized usingmethods known in the art such as binding a GST-NIPA-1 fusion protein toa polymeric bead containing glutathione. A chimeric gene encoding a GSTfusion protein is constructed by fusing DNA encoding the polypeptide orpolypeptide fragment of interest to the DNA encoding the carboxylterminus of GST (See e.g., Smith et al., Gene 67:31 [1988]). The fusionconstruct is then transformed into a suitable expression system (e.g.,E. coli XA90) in which the expression of the GST fusion protein can beinduced with isopropyl-β-D-thiogalactopyranoside (IPTG). Induction withIPTG should yield the fusion protein as a major constituent of soluble,cellular proteins. The fusion proteins can be purified by methods knownto those skilled in the art, including purification by glutathioneaffinity chromatography. Binding of the candidate compound to theproteins or protein fragments is correlated with the ability of thecompound to disrupt the signal transduction pathway and thus regulateNIPA-1 physiological effects (e.g., spastic paraplegia).

In another screening method, one of the components of the NIPA-1/bindingpartner signaling system is immobilized. Polypeptides can be immobilizedusing methods known in the art, such as adsorption onto a plasticmicrotiter plate or specific binding of a GST-fusion protein to apolymeric bead containing glutathione. For example, in some embodiments,GST-NIPA-1 is bound to glutathione-Sepharose beads. The immobilizedpeptide is then contacted with another peptide with which it is capableof binding in the presence and absence of a candidate compound. Unboundpeptide is then removed and the complex solubilized and analyzed todetermine the amount of bound labeled peptide. A decrease in binding isan indication that the candidate compound inhibits the interaction ofNIPA-1 with the other peptide. A variation of this method allows for thescreening of compounds that are capable of disrupting apreviously-formed protein/protein complex. For example, in someembodiments a complex comprising a NIPA-1 or a NIPA-1 fragment bound toanother peptide is immobilized as described above and contacted with acandidate compound. The dissolution of the complex by the candidatecompound correlates with the ability of the compound to disrupt orinhibit the interaction between NIPA-1 and the other peptide.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to NIPA-1 peptides and isdescribed in detail in WO 84/03564, incorporated herein by reference.Briefly, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are then reacted with NIPA-1peptides and washed. Bound NIPA-1 peptides are then detected by methodswell known in the art.

Another technique uses NIPA-1 antibodies, generated as discussed above.Such antibodies are capable of specifically binding to NIPA-1 peptidesand compete with a test compound for binding to NIPA-1. In this manner,the antibodies can be used to detect the presence of any peptide thatshares one or more antigenic determinants of a NIPA-1 peptide.

The present invention contemplates many other means of screeningcompounds. The examples provided above are presented merely toillustrate a range of techniques available. One of ordinary skill in theart will appreciate that many other screening methods can be used.

In particular, the present invention contemplates the use of cell linestransfected with NIPA-1 genes and variants thereof for screeningcompounds for activity, and in particular to high throughput screeningof compounds from combinatorial libraries (e.g., libraries containinggreater than 10⁴ compounds). The cell lines of the present invention canbe used in a variety of screening methods. In some embodiments, thecells can be used in second messenger assays that monitor signaltransduction following activation of cell-surface receptors. In otherembodiments, the cells can be used in reporter gene assays that monitorcellular responses at the transcription/translation level. In stillfurther embodiments, the cells can be used in cell proliferation assaysto monitor the overall growth/no growth response of cells to externalstimuli.

In second messenger assays, the host cells are preferably transfected asdescribed above with vectors encoding NIPA-1 or variants or mutantsthereof. The host cells are then treated with a compound or plurality ofcompounds (e.g., from a combinatorial library) and assayed for thepresence or absence of a response. It is contemplated that at least someof the compounds in the combinatorial library can serve as agonists,antagonists, activators, or inhibitors of the protein or proteinsencoded by the vectors. It is also contemplated that at least some ofthe compounds in the combinatorial library can serve as agonists,antagonists, activators, or inhibitors of protein acting upstream ordownstream of the protein encoded by the vector in a signal transductionpathway.

In some embodiments, the second messenger assays measure fluorescentsignals from reporter molecules that respond to intracellular changes(e.g., Ca²⁺ concentration, membrane potential, pH, IP₃, cAMP,arachidonic acid release) due to stimulation of membrane receptors andion channels (e.g., ligand gated ion channels; see Denyer et al., DrugDiscov. Today 3:323 [1998]; and Gonzales et al., Drug. Discov. Today4:431-39 [1999]). Examples of reporter molecules include, but are notlimited to, FRET (florescence resonance energy transfer) systems (e.g.,Cuo-lipids and oxonols, EDAN/DABCYL), calcium sensitive indicators(e.g., Fluo-3, FURA 2, INDO 1, and FLUO3/AM, BAPTA AM),chloride-sensitive indicators (e.g., SPQ, SPA), potassium-sensitiveindicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI), andpH sensitive indicators (e.g., BCECF).

In general, the host cells are loaded with the indicator prior toexposure to the compound. Responses of the host cells to treatment withthe compounds can be detected by methods known in the art, including,but not limited to, fluorescence microscopy, confocal microscopy (e.g.,FCS systems), flow cytometry, microfluidic devices, FLIPR systems (See,e.g., Schroeder and Neagle, J. Biomol. Screening 1:75 [1996]), andplate-reading systems. In some preferred embodiments, the response(e.g., increase in fluorescent intensity) caused by compound of unknownactivity is compared to the response generated by a known agonist andexpressed as a percentage of the maximal response of the known agonist.The maximum response caused by a known agonist is defined as a 100%response. Likewise, the maximal response recorded after addition of anagonist to a sample containing a known or test antagonist is detectablylower than the 100% response.

The cells are also useful in reporter gene assays. Reporter gene assaysinvolve the use of host cells transfected with vectors encoding anucleic acid comprising transcriptional control elements of a targetgene (i.e., a gene that controls the biological expression and functionof a disease target) spliced to a coding sequence for a reporter gene.Therefore, activation of the target gene results in activation of thereporter gene product. In some embodiments, the reporter gene constructcomprises the 5′ regulatory region (e.g., promoters and/or enhancers) ofa protein whose expression is controlled by NIPA-1 in operableassociation with a reporter gene. Examples of reporter genes finding usein the present invention include, but are not limited to,chloramphenicol transferase, alkaline phosphatase, firefly and bacterialluciferases, β-galactosidase, β-lactamase, and green fluorescentprotein. The production of these proteins, with the exception of greenfluorescent protein, is detected through the use of chemiluminescent,colorimetric, or bioluminescent products of specific substrates (e.g.,X-gal and luciferin). Comparisons between compounds of known and unknownactivities may be conducted as described above.

Specifically, the present invention provides screening methods foridentifying modulators, i.e., candidate or test compounds or agents(e.g., proteins, peptides, peptidomimetics, peptoids, small molecules orother drugs) which bind to a NIPA-1 of the present invention, have aninhibitory (or stimulatory) effect on, for example, NIPA-1 expression orNIPA-1 activity, or have a stimulatory or inhibitory effect on, forexample, the expression or activity of a NIPA-1 substrate. Compoundsthus identified can be used to modulate the activity of target geneproducts (e.g., NIPA-1 genes) either directly or indirectly in atherapeutic protocol, to elaborate the biological function of the targetgene product, or to identify compounds that disrupt normal target geneinteractions. Compounds, which stimulate the activity of a variantNIPA-1 or mimic the activity of a non-functional variant areparticularly useful in the treatment of neurological disorders (e.g.,HSP).

In one embodiment, the invention provides assays for screening candidateor test compounds that are substrates of a NIPA-1 protein or polypeptideor a biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compounds thatbind to or modulate the activity of a NIPA-1 protein or polypeptide or abiologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including biological libraries; peptoid libraries (libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone, which are resistant to enzymatic degradation butwhich nevertheless remain bioactive; see, e.g., Zuckennann et al., J.Med. Chem. 37: 2678-85 [1994]); spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary and peptoid library approaches are preferred for use withpeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84[1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores(U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids(Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage(Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406[1990]; Cwirla et al., Proc. NatI. Acad. Sci. 87:6378-6382 [1990];Felici, J. Mol. Biol. 222:301 [1991]).

In one embodiment, an assay is a cell-based assay in which a cell thatexpresses a NIPA-1 protein or biologically active portion thereof iscontacted with a test compound, and the ability of the test compound tomodulate a NIPA-1's activity is determined. Determining the ability ofthe test compound to modulate NIPA-1 activity can be accomplished bymonitoring, for example, changes in enzymatic activity. The cell, forexample, can be of mammalian origin.

The ability of the test compound to modulate NIPA-1 binding to acompound, e.g., a NIPA-1 substrate, can also be evaluated. This can beaccomplished, for example, by coupling the compound, e.g., thesubstrate, with a radioisotope or enzymatic label such that binding ofthe compound, e.g., the substrate, to a NIPA-1 can be determined bydetecting the labeled compound, e.g., substrate, in a complex.

Alternatively, a NIPA-1 is coupled with a radioisotope or enzymaticlabel to monitor the ability of a test compound to modulate NIPA-1binding to a NIPA-1 substrate in a complex. For example, compounds(e.g., substrates) can be labeled with ¹²⁵I, ³⁵S ¹⁴C or ³H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radioemmission or by scintillation counting. Alternatively, compoundscan be enzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

The ability of a compound (e.g., a NIPA-1 substrate) to interact with aNIPA-1 with or without the labeling of any of the interactants can beevaluated. For example, a microphysiorneter can be used to detect theinteraction of a compound with a NIPA-1 without the labeling of eitherthe compound or the NIPA-1 (McConnell et al. Science 257:1906-1912[1992]). As used herein, a “microphysiometer” (e.g., Cytosensor) is ananalytical instrument that measures the rate at which a cell acidifiesits environment using a light-addressable potentiometric sensor (LAPS).Changes in this acidification rate can be used as an indicator of theinteraction between a compound and a NIPA-1 polypeptide.

In yet another embodiment, a cell-free assay is provided in which aNIPA-1 protein or biologically active portion thereof is contacted witha test compound and the ability of the test compound to bind to theNIPA-1 protein or biologically active portion thereof is evaluated.Preferred biologically active portions of NIPA-1 proteins to be used inassays of the present invention include fragments that participate ininteractions with substrates or other proteins, e.g., fragments withhigh surface probability scores.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FRET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No.4,968,103; each of which is herein incorporated by reference). Afluorophore label is selected such that a first donor molecule's emittedfluorescent energy will be absorbed by a fluorescent label on a second,‘acceptor’ molecule, which in turn is able to fluoresce due to theabsorbed energy.

Alternately, the ‘donor’ protein molecule may simply utilize the naturalfluorescent energy of tryptophan residues. Labels are chosen that emitdifferent wavelengths of light, such that the ‘acceptor’ molecule labelmay be differentiated from that of the ‘donor’. Since the efficiency ofenergy transfer between the labels is related to the distance separatingthe molecules, the spatial relationship between the molecules can beassessed. In a situation in which binding occurs between the molecules,the fluorescent emission of the ‘acceptor’ molecule label in 1 5 theassay should be maximal. An FRET binding event can be convenientlymeasured through standard fluorometric detection means well known in theart (e.g., using a fluorimeter).

In another embodiment, determining the ability of a NIPA-1 protein tobind to a target molecule can be accomplished using real-timeBiomolecular Interaction Analysis (BIA) (see, e.g., Sjolander andUrbaniczky, Anal. Chem. 63:2338-2345 [1991] and Szabo et al. Curr. Opin.Struct. Biol. 5:699-705 [1995]). “Surface plasmon resonance” or “BIA”detects biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcore). Changes in the mass at the bindingsurface (indicative of a binding event) result in alterations of therefractive index of light near the surface (the optical phenomenon ofsurface plasmon resonance (SPR)), resulting in a detectable signal thatcan be used as an indication of real-time reactions between biologicalmolecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize a NIPA-1 protein, an anti-NIPA-1antibody or its target molecule to facilitate separation of complexedfrom non-complexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound to aNIPA-1 protein, or interaction of a NIPA-1 protein with a targetmolecule in the presence and absence of a candidate compound, can beaccomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase-NIPA-1fusion proteins or glutathione-S-transferase/target fusion proteins canbe adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione-derivatized microtiter plates, which are thencombined with the test compound or the test compound and either thenon-adsorbed target protein or NIPA-1 protein, and the mixture incubatedunder conditions conducive for complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above.

Alternatively, the complexes can be dissociated from the matrix, and thelevel of NIPA-1 binding or activity determined using standardtechniques. Other techniques for immobilizing either a NIPA-1 protein ora target molecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated NIPA-1 protein or target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques knownin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, EL),and immobilized in the wells of streptavidin-coated 96 well plates(Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-IgG antibody).

This assay is performed utilizing antibodies reactive with NIPA-1protein or target molecules but which do not interfere with binding ofthe NIPA-1 protein to its target molecule. Such antibodies can bederivatized to the wells of the plate, and unbound target or NIPA-1protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immuNIPA-1 etection of complexesusing antibodies reactive with the NIPA-1 protein or target molecule, aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the NIPA-1 protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including, butnot limited to: differential centrifugation (see, for example, Rivas andMinton, Trends Biochem Sci 18:284-7 [1993]); chromatography (gelfiltration chromatography, ion-exchange chromatography); electrophoresis(see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology1999, J. Wiley: New York.); and immunoprecipitation (see, for example,Ausubel et al., eds. Current Protocols in Molecular Biology 1999, J.Wiley: New York). Such resins and chromatographic techniques are knownto one skilled in the art (See e.g., Heegaard J. Mol. Recognit 11:141-8[1998]; Hageand Tweed J. Chromatogr. Biomed. Sci. Appl 699:499-525[1997]). Further, fluorescence energy transfer may also be convenientlyutilized, as described herein, to detect binding without furtherpurification of the complex from solution.

The assay can include contacting the NIPA-1 protein or biologicallyactive portion thereof with a known compound that binds the NIPA-1 toform an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a NIPA-1 protein, wherein determining the ability of the testcompound to interact with a NIPA-1 protein includes determining theability of the test compound to preferentially bind to NIPA-1 orbiologically active portion thereof, or to modulate the activity of atarget molecule, as compared to the known compound.

To the extent that a NIPA-1 can, in vivo, interact with one or morecellular or extracellular macromolecules, such as proteins, inhibitorsof such an interaction are useful. A homogeneous assay can be used canbe used to identify inhibitors.

For example, a preformed complex of the target gene product and theinteractive cellular or extracellular binding partner product isprepared such that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496, hereinincorporated by reference, that utilizes this approach forimmunoassays). The addition of a test substance that competes with anddisplaces one of the species from the preformed complex will result inthe generation of a signal above background. In this way, testsubstances that disrupt target gene product-binding partner interactioncan be identified. Alternatively, a NIPA-1 protein can be used as a“bait protein” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al., Cell 72:223-232 [1993]; Maduraet al., J. Biol. Chem. 268.12046-12054 [1993]; Bartel et al.,Biotechniques 14:920-924 [1993]; Iwabuchi et al., Oncogene 8:1693-1696[1993]; and Brent WO 94/10300; each of which is herein incorporated byreference), to identify other proteins, that bind to or interact with aNIPA-1 (“NIPA-1-binding proteins” or “NIPA-1-bp”) and are involved inNIPA-1 activity. Such NIPA-1-bps can be activators or inhibitors ofsignals by the NIPA-1 proteins or targets as, for example, downstreamelements of a NIPA-1-mediated signaling pathway.

Modulators of NIPA-1 expression can also be identified. For example, acell or cell free mixture is contacted with a candidate compound and theexpression of a NIPA-1 mRNA or protein evaluated relative to the levelof expression of the NIPA-1 mRNA or protein in the absence of thecandidate compound. When expression of the NIPA-1 mRNA or protein isgreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of a NIPA-1 mRNA orprotein expression. Alternatively, when expression of NIPA-1 mRNA orprotein is less (i.e., statistically significantly less) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as an inhibitor of NIPA-1 mRNA or protein expression. Thelevel of NIPA-1 mRNA or protein expression can be determined by methodsdescribed herein for detecting NIPA-1 mRNA or protein.

A modulating agent can be identified using a cell-based or a cell freeassay, and the ability of the agent to modulate the activity of a NIPA-1protein can be confirmed in vivo, e.g., in an animal such as an animalmodel for a disease (e.g., an animal with HSP).

B. Therapeutic Agents

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a NIPA-1 modulating agent or mimetic, a NIPA-1 specific antibody,or a NIPA-1-binding partner) in an appropriate animal model (such asthose described herein) to determine the efficacy, toxicity, sideeffects, or mechanism of action, of treatment with such an agent.Furthermore, as described above, novel agents identified by theabove-described screening assays can be, e.g., used for treatments ofneurological disorders (e.g., including, but not limited to, HSP). Insome embodiments, the agents are NIPA-1 ligands or ligand analogs (e.g.,identified using the drug screening methods described above).

IX. Pharmaceutical Compositions Containing NIPA-1 Nucleic Acid,Peptides, and Analogs

The present invention further provides pharmaceutical compositions whichmay comprise all or portions of NIPA-1 polynucleotide sequences, NIPA-1polypeptides, inhibitors or antagonists of NIPA-1 bioactivity, includingantibodies, alone or in combination with at least one other agent, suchas a stabilizing compound, and may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water.

The methods of the present invention find use in treating diseases oraltering physiological states characterized by mutant NIPA-1 alleles(e.g., spastic paraplegia diseases). Peptides can be administered to thepatient intravenously in a pharmaceutically acceptable carrier such asphysiological saline. Standard methods for intracellular delivery ofpeptides can be used (e.g., delivery via liposome). Such methods arewell known to those of ordinary skill in the art. The formulations ofthis invention are useful for parenteral administration, such asintravenous, subcutaneous, intramuscular, and intraperitoneal.Therapeutic administration of a polypeptide intracellularly can also beaccomplished using gene therapy as described above.

As is well known in the medical arts, dosages for any one patientdepends upon many factors, including the patient's size, body surfacearea, age, the particular compound to be administered, sex, time androute of administration, general health, and interaction with otherdrugs being concurrently administered.

Accordingly, in some embodiments of the present invention, NIPA-1nucleotide and NIPA-1 amino acid sequences can be administered to apatient alone, or in combination with other nucleotide sequences, drugsor hormones or in pharmaceutical compositions where it is mixed withexcipient(s) or other pharmaceutically acceptable carriers. In oneembodiment of the present invention, the pharmaceutically acceptablecarrier is pharmaceutically inert. In another embodiment of the presentinvention, NIPA-1 polynucleotide sequences or NIPA-1 amino acidsequences may be administered alone to individuals subject to orsuffering from a disease.

Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration.

For injection, the pharmaceutical compositions of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. For tissue or cellular administration,penetrants appropriate to the particular barrier to be permeated areused in the formulation. Such penetrants are generally known in the art.

In other embodiments, the pharmaceutical compositions of the presentinvention can be formulated using pharmaceutically acceptable carrierswell known in the art in dosages suitable for oral administration. Suchcarriers enable the pharmaceutical compositions to be formulated astablets, pills, capsules, liquids, gels, syrups, slurries, suspensionsand the like, for oral or nasal ingestion by a patient to be treated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. For example, aneffective amount of NIPA-1 may be that amount that suppresses spasticparaplegia related symptoms. Determination of effective amounts is wellwithin the capability of those skilled in the art, especially in lightof the disclosure provided herein.

In addition to the active ingredients these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known (e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes).

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are carbohydrate or protein fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; starch from corn,wheat, rice, potato, etc; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; and proteins such as gelatin andcollagen. If desired, disintegrating or solubilizing agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, (i.e., dosage).

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients mixed with a filler orbinders such as lactose or starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier may be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. For polynucleotide or amino acid sequences of NIPA-1,conditions indicated on the label may include treatment of conditionrelated to spastic paraplegia diseases.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with bufferprior to use.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. Then, preferably, dosage can be formulated in animalmodels (particularly murine models) to achieve a desirable circulatingconcentration range that adjusts NIPA-1 levels.

A therapeutically effective dose refers to that amount of NIPA-1 thatameliorates symptoms of the disease state. Toxicity and therapeuticefficacy of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds thatexhibit large therapeutic indices are preferred. The data obtained fromthese cell culture assays and additional animal studies can be used informulating a range of dosage for human use. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage varieswithin this range depending upon the dosage form employed, sensitivityof the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of thepatient to be treated. Dosage and administration are adjusted to providesufficient levels of the active moiety or to maintain the desiredeffect. Additional factors which may be taken into account include theseverity of the disease state; age, weight, and gender of the patient;diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance/response to therapy. Long actingpharmaceutical compositions might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.01 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature (See, U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212,all of which are herein incorporated by reference). Those skilled in theart will employ different formulations for NIPA-1 than for theinhibitors of NIPA-1. Administration to the bone marrow may necessitatedelivery in a manner different from intravenous injections.

X. RNA Interference (RNAi)

RNAi represents an evolutionary conserved cellular defense forcontrolling the expression of foreign genes in most eukaryotes,including humans. RNAi is triggered by double-stranded RNA (dsRNA) andcauses sequence-specific mRNA degradation of single-stranded target RNAshomologous in response to dsRNA. The mediators of mRNA degradation aresmall interfering RNA duplexes (siRNAs), which are normally producedfrom long dsRNA by enzymatic cleavage in the cell. siRNAs are generallyapproximately twenty-one nucleotides in length (e.g. 21-23 nucleotidesin length), and have a base-paired structure characterized by twonucleotide 3′-overhangs. Following the introduction of a small RNA, orRNAi, into the cell, it is believed the sequence is delivered to anenzyme complex called RISC (RNA-induced silencing complex). RISCrecognizes the target and cleaves it with an endonuclease. It is notedthat if larger RNA sequences are delivered to a cell, RNase III enzyme(Dicer) converts longer dsRNA into 21-23 nt ds siRNA fragments.

Chemically synthesized siRNAs have become powerful reagents forgenome-wide analysis of mammalian gene function in cultured somaticcells. Beyond their value for validation of gene function, siRNAs alsohold great potential as gene-specific therapeutic agents (Tuschl andBorkhardt, Molecular Intervent. 2002; 2(3):158-67, herein incorporatedby reference).

The transfection of siRNAs into animal cells results in the potent,long-lasting post-transcriptional silencing of specific genes (Caplen etal, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al., Nature.2001; 411:494-8; Elbashir et al., Genes Dev. 2001; 15: 188-200; andElbashir et al., EMBO J. 2001; 20: 6877-88, all of which are hereinincorporated by reference). Methods and compositions for performing RNAiwith siRNAs are described, for example, in U.S. Pat. No. 6,506,559,herein incorporated by reference.

siRNAs are extraordinarily effective at lowering the amounts of targetedRNA, and by extension proteins, frequently to undetectable levels. Thesilencing effect can last several months, and is extraordinarilyspecific, because one nucleotide mismatch between the target RNA and thecentral region of the siRNA is frequently sufficient to preventsilencing Brummelkamp et al, Science 2002; 296:550-3; and Holen et al,Nucleic Acids Res. 2002; 30:1757-66, both of which are hereinincorporated by reference.

XI. RNAi for NIPA-1

As discussed above, the present invention provides RNAi for inhibitingthe expression of the NIPA-1 polypeptide in cells. Preferably,inhibition of the level of NIPA-1 expression in cells prevents and/orreduces the symptoms of HSP.

A. Designing and Testing RNAi for NIPA-1

In order to design siRNAs for NIPA-1 (e.g. that target NIPA-1 mRNA)software design tools are available in the art (e.g. on the internet).For example, Oligoengine's web page has one such design tool that findsRNAi candidates based on Elbashir's (Elbashir et al, Methods 2002; 26:199-213, herein incorporated by reference) criteria. Other design toolsmay also be used, such as the Cenix Bioscience design tool offered byAmbion. In addition, there is also the Si2 silencing duplex offered byOligoengine.

There are also RNA folding software programs available that allow one todetermine if the mRNA has a tendency to fold on its own and form a“hair-pin” (which in the case of dsRNAi is not as desirable since onegoal is to have the RNAi attach to the mRNA and not itself). Onepreferred configuration is an open configuration with three or lessbonds. Generally, a positive delta G is desirable to show that it wouldnot tend to fold on itself spontaneously.

siRNA candidate molecules that are generated can be, for example,screened in an animal model of HSP for the quantitative evaluation ofNIPA-1 expression in vivo using similar techniques as described above.

B. Expression Cassettes

NIPA-1 specific siRNAs of the present invention may be synthesizedchemically. Chemical synthesis can be achieved by any method known ordiscovered in the art. Alternatively, NIPA-1 specific siRNAs of thepresent invention may be synthesized by methods which comprise synthesisby transcription. In some embodiments, transcription is in vitro, asfrom a DNA template and bacteriophage RNA polymerase promoter, in otherembodiments, synthesis is in vivo, as from a gene and a promoter.Separate-stranded duplex siRNA, where the two strands are synthesizedseparately and annealed, can also be synthesized chemically by anymethod known or discovered in the art. Alternatively, ds siRNA aresynthesized by methods which comprise synthesis by transcription. Insome embodiments, the two strands of the double-stranded region of asiRNA are expressed separately by two different expression cassettes,either in vitro (e.g., in a transcription system) or in vivo in a hostcell, and then brought together to form a duplex.

Thus, in another aspect, the present invention provides a compositioncomprising an expression cassette comprising a promoter and a gene thatencodes a siRNA specific for NIPA-1. In some embodiments, thetranscribed siRNA forms a single strand of a separate-stranded duplex(or double-stranded, or ds) siRNA of about 18 to 25 base pairs long;thus, formation of ds siRNA requires transcription of each of the twodifferent strands of a ds siRNA. The term “gene” in the expressioncassette refers to a nucleic acid sequence that comprises codingsequences necessary for the production of a siRNA. Thus, a gene includesbut is not limited to coding sequences for a strand of a ds siRNA.

Generally, a DNA expression cassette comprises a chemically synthesizedor recombinant DNA molecule containing at least one gene, or desiredcoding sequence for a single strand of a ds siRNA, and appropriatenucleic acid sequences necessary for the expression of the operablylinked coding sequence, either in vitro or in vivo. Expression in vitromay include expression in transcription systems and intranscription/translation systems. Expression in vivo may includeexpression in a particular host cell and/or organism. Nucleic acidsequences necessary for expression in a prokaryotic cell or in aprokaryotic in vitro expression system are well known and usuallyinclude a promoter, an operator, and a ribosome binding site, oftenalong with other sequences. Eukaryotic in vitro transcription systemsand cells are known to utilize promoters, enhancers, and termination andpolyadenylation signals. Nucleic acid sequences necessary for expressionvia bacterial RNA polymerases (such as T3, T7, and SP6), referred to asa transcription template in the art, include a template DNA strand whichhas a polymerase promoter region followed by the complement of the RNAsequence desired (or the coding sequence or gene for the siRNA). Inorder to create a transcription template, a complementary strand isannealed to the promoter portion of the template strand.

In any of the expression cassettes described above, the gene may encodea transcript that contains at least one cleavage site, such that whencleaved results in at least two cleavage products. Such products caninclude the two opposite strands of a ds siRNA. In an expression systemfor expression in a eukaryotic cell, the promoter may be constitutive orinducible; the promoter may also be tissue or organ specific (e.g.specific to the eye), or specific to a developmental phase. Preferably,the promoter is positioned 5′ to the transcribed region. Other promotersare also contemplated; such promoters include other polymerase IIIpromoters and microRNA promoters.

Preferably, a eukaryotic expression cassette further comprises atranscription termination signal suitable for use with the promoter; forexample, when the promoter is recognized by RNA polymerase III, thetermination signal is an RNA polymerase III termination signal. Thecassette may also include sites for stable integration into a host cellgenome.

C. Vectors

In other aspects of the present invention, the compositions comprise avector comprising a gene encoding an siRNA specific for NIPA-1 orpreferably at least one expression cassette comprising a promoter and agene which encodes a sequence necessary for the production of a siRNAspecific for NIPA-1 (an siRNA gene). The vectors may further comprisemarker genes, reporter genes, selection genes, or genes of interest,such as experimental genes. Vectors of the present invention includecloning vectors and expression vectors. Expression vectors may be usedin in vitro transcription/translation systems, as well as in in vivo ina host cell. Expression vectors used in vivo in a host cell may betransfected into a host cell, either transiently, or stably. Thus, avector may also include sites for stable integration into a host cellgenome.

In some embodiments, it is useful to clone a siRNA gene downstream of abacteriophage RNA polymerase promoter into a multicopy plasmid. Avariety of transcription vectors containing bacteriophage RNA polymerasepromoters (such as T7 promoters) are available. Alternatively, DNAsynthesis can be used to add a bacteriophage RNA polymerase promoterupstream of a siRNA coding sequence. The cloned plasmid DNA, linearizedwith a restriction enzyme, can then be used as a transcription template(See for example Milligan, J F and Uhlenbeck, O C (1989) Methods inEnzymology 180: 51-64).

In other embodiments of the present invention, vectors include, but arenot limited to, chromosomal, nonchromosomal and synthetic DNA sequences(e.g., derivatives of viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies). It is contemplated that any vector may be usedas long as it is expressed in the appropriate system (either in vitro orin vivo) and viable in the host when used in vivo; these two criteriaare sufficient for transient transfection. For stable transfection, thevector is also replicable in the host.

Large numbers of suitable vectors are known to those of skill in theart, and are commercially available. In some embodiments of the presentinvention, mammalian expression vectors comprise an origin ofreplication, suitable promoters and enhancers, and also any necessaryribosome binding sites, polyadenylation sites, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences. In other embodiments, DNA sequences derivedfrom the SV40 splice, and polyadenylation sites may be used to providethe required non-transcribed genetic elements.

In certain embodiments of the present invention, a gene sequence in anexpression vector which is not part of an expression cassette comprisinga siRNA gene (specific for NIPA-1) is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. In some embodiments, the gene sequence is a marker gene or aselection gene. Promoters useful in the present invention include, butare not limited to, the cytomegalovirus (CMV) immediate early, herpessimplex virus (HSV) thymidine kinase, and mouse metallothioneinpromoters and other promoters known to control expression of gene inmammalian cells or their viruses. In other embodiments of the presentinvention, recombinant expression vectors include origins of replicationand selectable markers permitting transformation of the host cell (e.g.,dihydrofolate reductase or neomycin resistance for eukaryotic cellculture).

In some embodiments of the present invention, transcription of DNAencoding a gene is increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp that act on a promoter to increase its transcription.Enhancers useful in the present invention include, but are not limitedto, a cytomegalovirus early promoter enhancer, the polyoma enhancer onthe late side of the replication origin, and adenovirus enhancers.

Preferably the design of a vector is configured to deliver the RNAi formore permanent inhibition. For example the pSilencer siRNA expressionvector offered by Ambion, the pSuper RNAi system offered by Oligoengine,and the GneSilencer System offered by IMGENEX. These are all plasmidvector based RNAis. BD Biosciences offer the RNAi-Ready pSIREN Vectors,that allow both a Plasmid-based vectors and an Adenoviral or aRetroviral delivery formats. Ambion is expected to release an adenoviralvector for siRNA shortly. For the design of a vector there is nolimitation regarding the folding pattern since there is no concernregarding the formation of a hairpin or at least there are no studiesthat found any difference in performance related to the mRNA foldingpattern. Therefore, SEQ ID NOS: 1 and 2, for example, may be used within a Vector (both Plasmid and Viral) delivery systems.

It is noted that Ambion offers a design tool for a vector on their webpage, and BD Biosciences offers a manual for the design of a vector,both of which are useful for designing vectors for siRNA.

D. Transfecting Cells

In yet other aspects, the present invention provides compositionscomprising cells transfected by an expression cassette of the presentinvention as described above, or by a vector of the present invention,where the vector comprises an expression cassette (or simply the siRNAgene) of the present invention, as described above. In some embodimentsof the present invention, the host cell is a mammalian cell. Atransfected cell may be a cultured cell or a tissue, organ, ororganismal cell. Specific examples of cultured host cells include, butare not limited to, Chinese hamster ovary (CHO) cells, COS-7 lines ofmonkey kidney fibroblasts, 293T, C127, 3T3, HeLa, and BHK cell lines.Specific examples of host cells in vivo include tumor tissue and eyetissue.

The cells may be transfected transiently or stably (e.g. DNA expressingthe siRNA is stably integrated and expressed by the host cell's genome).The cells may also be transfected with an expression cassette of thepresent invention, or they are transfected with an expression vector ofthe present invention. In some embodiments, transfected cells arecultured mammalian cells, preferably human cells. In other embodiments,they are tissue, organ, or organismal cells.

In the present invention, cells to be transfected in vitro are typicallycultured prior to transfection according to methods which are well knownin the art, as for example by the preferred methods as defined by theAmerican Tissue Culture Collection. In certain embodiments of thepresent invention, cells are transfected with siRNAs that aresynthesized exogenously (or in vitro, as by chemical methods or in vitrotranscription methods), or they are transfected with expressioncassettes or vectors, which express siRNAs within the transfected cell.

In some embodiments, cells are transfected with siRNAs by any methodknown or discovered in the art which allows a cell to take up exogenousRNA and remain viable. Non-limiting examples include electroporation,microinjection, transduction, cell fusion, DEAE dextran, calciumphosphate precipitation, use of a gene gun, osmotic shock, temperatureshock, and electroporation, and pressure treatment. In alternative,embodiments, the siRNAs are introduced in vivo by lipofection, as hasbeen reported (as, for example, by Elbashir et al. (2001) Nature 411:494-498, herein incorporated by reference).

In other embodiments expression cassettes or vectors comprising at leastone expression cassette are introduced into the desired host cells bymethods known in the art, including but not limited to transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (See e.g., Wu et al. (1992) J. Biol. Chem.,267:963; Wu and Wu (1988) J. Biol. Chem., 263:14621; and Williams et al.(1991) Proc. Natl. Acad. Sci. USA 88:272). Receptor-mediated DNAdelivery approaches are also used (Curiel et al. (1992) Hum. Gene Ther.,3:147; and Wu and Wu (1987) J. Biol. Chem., 262:4429). In someembodiments, various methods are used to enhance transfection of thecells. These methods include but are not limited to osmotic shock,temperature shock, and electroporation, and pressure treatment.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Syntheticcationic lipids designed to limit the difficulties and dangersencountered with liposome mediated transfection can be used to prepareliposomes for in vivo transfection of a gene encoding a marker. The useof cationic lipids may promote encapsulation of negatively chargednucleic acids, and also promote fusion with negatively charged cellmembranes. Particularly useful lipid compounds and compositions fortransfer of nucleic acids are described in WO95/18863 and WO96/17823,and in U.S. Pat. No. 5,459,127, herein incorporated by reference. Othermolecules are also useful for facilitating transfection of a nucleicacid in vivo, such as a cationic oligopeptide (e.g., WO95/21931),peptides derived from DNA binding proteins (e.g., WO96/25508), or acationic polymer (e.g., WO95/21931).

It is also possible to introduce a sequence encoding a siRNA in vivo asa naked DNA, either as an expression cassette or as a vector. Methodsfor formulating and administering naked DNA to mammalian muscle tissueare disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of whichare herein incorporated by reference.

Stable transfection typically requires the presence of a selectablemarker in the vector used for transfection. Transfected cells are thensubjected to a selection procedure. Generally, selection involvesgrowing the cells in a toxic substance, such as G418 or Hygromycin B,such that only those cells expressing a transfected marker geneconferring resistance to the toxic substance upon the transfected cellsurvive and grow. Such selection techniques are well known in the art.Typical selectable markers are well known, and include genes encodingresistance to G418 or hygromycin B.

In preferred embodiments, the transfecting agent is OLIGOFECTAMINE.OLIGOFECTAMINE is a lipid based transfection reagent. Additional exampleof lipid based transfection reagents that were designed for thetransfection of dsRNAis are the Transit-TKO reagent which is provided byMirus (Madison, Wis.) and the jetSI which was introduced byPolyplus-trasfection SAS. In addition, the Silencer siRNA TransfectionKit provided by Ambion's includes siPORT Amine and siPORT Lipidtransfection agents. Roche offers the Fugene 6 transfection reagentsthat are also lipid based. There is an option to use electroporation incell culture. Preferably a plasmid vector delivery system is transfectedinto the cell with OLIGOFECTAMINE provided by Invitrogen or with siPORTXP-1 transfection agent provided by Ambion.

In certain embodiments, certain chemical modifications of the dsRNAissuch as changing the lipophilicity of the molecule may be employed(e.g., attachment of lipophilic residues at the 3′ termini of thedsRNA). Delivery of dsRNAs into organisms may also be achieved withmethods previously developed for the application of antisenseoligonucleotides such as injection of liposomes-encapsulated molecules.

E. Kits

The present invention also provides kits comprising at least oneexpression cassette comprising a siRNA gene specific for NIPA-1. In someaspects, a transcript from the expression cassette forms a doublestranded siRNA of about 18 to 25 base pairs long. In other embodiments,the expression cassette is contained within a vector, as describedabove, where the vector can be used in in vitro transcription ortranscription/translation systems, or used in vivo to transfect cells,either transiently or stably.

In other aspects, the kit comprises at least two expression cassettes,each of which comprises a siRNA gene, such that at least one geneencodes one strand of a siRNA that combines with a strand encoded by asecond cassette to form a ds siRNA; the ds siRNA so produced is any ofthe embodiments described above. These cassettes may comprise a promoterand a sequence encoding one strand of a ds siRNA. In some furtherembodiments, the two expression cassettes are present in a singlevector; in other embodiments, the two expression cassettes are presentin two different vectors. A vector with at least one expressioncassette, or two different vectors, each comprising a single expressioncassette, can be used in in vitro transcription ortranscription/translation systems, or used in vivo to transfect cells,either transiently or stably.

In yet other aspects, the kit comprises at least one expressioncassettes which comprises a gene which encodes two separate strands of ads siRNA and a processing site between the sequences encoding eachstrand such that, when the gene is transcribed, the transcript isprocessed, such as by cleavage, to result in two separate strands whichcan combine to form a ds siRNA, as described above.

In some embodiments, the present invention provides kits comprising; a)a composition comprising small interfering RNA duplexes (siRNAs)configured to inhibit expression of the NIPA-1 protein, and b) printedmaterial with instructions for employing the composition for treating atarget cell expressing NIPA-1 protein via expression of NIPA-1 mRNAunder conditions such that the NIPA-1 mRNA is cleaved or otherwisedisabled. In certain embodiments, the printed material comprisesinstructions for employing the composition for treating eye disease.

F. Generating NIPA-1 Specific siRNA

The present invention also provides methods of synthesizing siRNAsspecific for NIPA-1 (e.g. human NIPA-1) or specific for mutant or wildtype forms of NIPA-1. The siRNAs may be synthesized in vitro or in vivo.In vitro synthesis includes chemical synthesis and synthesis by in vitrotranscription. In vitro transcription is achieved in a transcriptionsystem, as from a bacteriophage RNA polymerase, or in atranscription/translation system, as from a eukaryotic RNA polymerase.In vivo synthesis occurs in a transfected host cell.

The siRNAs synthesized in vitro, either chemically or by transcription,are used to transfect cells. Therefore, the present invention alsoprovides methods of transfecting host cells with siRNAs synthesized invitro; in particular embodiments, the siRNAs are synthesized by in vitrotranscription. The present invention further provides methods ofsilencing the NIPA-1 gene in vivo by transfecting cells with siRNAssynthesized in vitro. In other methods, the siRNAs is expressed in vitroin a transcription/translation system from an expression cassette orexpression vector, along with an expression vector encoding andexpressing a reporter gene.

The present invention also provides methods of expressing siRNAs in vivoby transfecting cells with expression cassettes or vectors which directsynthesis of siRNAs in vivo. The present invention also provides methodsof silencing genes in vivo by transfecting cells with expressioncassettes or vectors that direct synthesis of siRNAs in vivo.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

ADHSP does not Result from Genetic Imprinting in Chromosome 15q11-13(SPG-6 Locus)

Ten ADHSP loci have been mapped, and four ADHSP genes have beenidentified—SPG4/spastin, SPG3A/atlastin, SPG13/chaperonin 60 andSPG10/KIF5A (Hazan et al, Nat Genet 23:296-303 (1999); Zhao et al, NatGenet 29:326-331 (2001); Hansen et al, Am J Hum Genet 70:1328-1332(2002); Reid et al, Am J Hum Genet 71:1189-1194 (2002)). Despite theseadvances, the molecular pathophysiology of the ADHSPs is unknown. Alocus for uncomplicated ADHSP is located in chromosome 15q (SPG6) (Finket al, Am J Hum Genet 56:188-92 (1995); Fink et al, Neurology 45:325-31(1995)).

The SPG6 locus extends 6.1 cM between D15S128 and the centromere(Rainier et al, Am J Hum Genet 67:91 (2000) (FIG. 2a ). This interval isinvolved in deletions that result in Prader-Willi syndrome (PWS) orAngelman syndrome (AS). PWS and AS are characterized by geneticimprinting (Nicholls and Knepper, Ann Rev Genomics Hum Genet 2:153-175(2001)). Studies conducted in the course of the present inventioninvolved analysis of a large kindred, ADHSP-ARK1 (FIG. 1b ), in whichADHSP was linked to the SPG6 locus. Analysis of the ADHSP-ARK1 kindredindicated no evidence of genetic imprinting (Fink et al, Neurology45:325-31 (1995)). The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, it is contemplated thatADHSP with the SPG-6 locus does not result from genetic imprinting.

A Mutation within the NIPA-1 Gene Causes ADHSP

In order to understand the molecular pathology surrounding ADHSP, fourunique, non-imprinted and highly evolutionarily conserved genes wereanalyzed. These genes mapped proximal of the imprinted domain and withinthe pericentromeric region of chromosome 15q (Chai et al, Am J Hum Genet(2003 in press)). These candidate genes included “non-imprinted inPrader-Willi/Angleman locus 1 (NIPA-1) (SEQ ID NO: 1) (NCBI-BK001020)and NIPA2 (NCBI-BK001120) (Chai et al, Am J Hum Genet (2003 in press)),GCP5 (NCBI-AF272884) (Murphy et al, Mol. Biol Cell 12:3340-3352 (2001))and CYFIP1 (NCBI-NM_014608) (Koybayashi et al, J Biol Chem 273:291-295(1998)); Schenk et al, Proc Natl Acad Sci (USA) 98:8844-8849 (2001)).

A nucleotide substitution at position 159 of the NIPA1 cDNA (159C>G;FIG. 1a ) was identified which resulted in an amino acid substitution atposition 45 of the NIPA-1 protein (T45R) in each affected subject (n=29)in ADHSP-ARK1 (FIG. 1b ). In contrast, each unaffected subject (n=29)had only C at this position (FIG. 1b ), which agrees with the knownhuman genomic sequence (NCBI-NT_024668). 105 control subjects(ascertained through the Elderly Subjects Program of the University ofMichigan Institute of Gerontology) were also examined. Each controlsubject had only C at position 159 of the NIPA1 cDNA.

Analysis of the coding sequence of the other three non-imprinted genes(GCP5, CYFIP1 and NIPA2) in two affected members of ADHSP-ARK1 andidentified no disease-specific mutations.

The NIPA1 coding sequence in affected probands from 62 ADHSP kindreds, 6probable autosomal recessive HSP kindreds, and 13 subjects with allsigns and symptoms of but no family history (“apparently sporadic”spastic paraplegia) were analyzed. Affected subjects in one unrelatedkindred (ADHSP-IRQ1; FIG. 1b ) had precisely the same NIPA1 mutation(159C>G; FIG. 1b ) as affected subjects in the ADHSP-ARK1 kindred.Unaffected subjects from ADHSP-IRQ1 kindred showed only the normalnucleotide (159C). Whereas the ADHSP-ARK1 kindred was linked to the SPG6locus (Fink et al, Am J Hum Genet 56:188-92 (1995)), the ADHSP-IRQ1kindred was too small for meaningful linkage analysis. Clinical featuresof the ADHSP-ARK1 affected individuals are typical of uncomplicated HSPof late-teenage to early-adult symptom onset (Fink et al, Neurology45:325-31 (1995)). Clinical features of ADHSP-IRQ1 were similar: onsetof insidiously progressive spastic weakness in both legs that began inlate teen-age years and was associated with urinary urgency and mildvibratory sensation impairment in the toes.

Kindreds ADHSP-ARK1 and ADHSP-IRQ1 are of Irish and Iraqi ancestry,respectively. Analysis of haplotypes for polymorphic markers linked tothis locus (D155541, D155542, D15S646, D15S817, D15S1021) showed noevidence of haplotype sharing between ADHSP-ARK1 and ADHSP-IRQ1kindreds. This indicates that these two ADHSP families are not closelyrelated and suggests that the same NIPA1 mutation arose independently inthese ADHSP kindreds.

The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, it is contemplated that ADHSP within theSPG-6 locus is caused by a mutation within the NIPA-1 gene.

ADHSP Operates Through a Mutation within the NIPA-1 Polypeptide

Disease-specific NIPA1 mutations in ADHSP-ARK1 and ADHSP-IRQ1 occur inNIPA1 exon 1 and change threonine to arginine at amino acid position 45(T45R) (FIG. 2b ). This amino acid is conserved in mouse, chicken andfish (zebrafish and Fugu) (Chai et al, Am J Hum Genet (2003 in press)).The present invention is not limited to a particular mechanism. Indeed,an understanding of the mechanism is not necessary to practice thepresent invention. Nonetheless, it is contemplated that thedisease-specific NIPA1 mutation changing threonine to arginine at aminoacid position 45 (T45R) occurs at the end of the first of ninetransmembrane domains in the NIPA-1 polypeptide (FIG. 2b ). NIPA-1 doesnot contain an AAA domain (as is present in spastin (Hazan et al, NatGenet 23:296-303 (1999)) or GTPase domain (as is present in atlastin(Zhao et al, Nat Genet 29:326-331 (2001)) or bear other homology togenes that cause other forms of HSP. The present invention is notlimited to a particular mechanism. Indeed, an understanding of themechanism is not necessary to practice the present invention.Nonetheless, it is contemplated that NIPA-1 functions as a receptor ortransporter. Many PWS or AS individuals have chromosome 15q class Ideletions that include NIPA1 (FIG. 2a ; (Chai et al, Am J Hum Genet(2003 in press)). The fact that such individuals do not exhibitprogressive spastic paraplegia shows that NIPA1 haploinsufficiency doesnot cause progressive spastic paraplegia. The present invention is notlimited to a particular mechanism. Indeed, an understanding of themechanism is not necessary to practice the present invention.Nonetheless, it is contemplated that the NIPA1 T45R missense mutationidentified in these ADHSP kindreds is pathogenic through a dominantnegative, gain-of-function mechanism.

NIPA1 mRNA is expressed constitutively at low levels with 2.2- and7.5-kb transcripts in all human tissues, but shows significantenrichment in the brain (FIG. 2c ). The latter expression pattern isfound throughout the central nervous system whereas spinal cord showsequal expression of the two NIPA1 mRNAs (FIG. 2c ). The alternative mRNAisoforms arise from alternative polyadenylation within NIPA1 exon 5, andequivalent expression patterns are found for mouse (Chai et al, Am J HumGenet (2003 in press)).

Discussion

Observations of the same NIPA-1 gene mutation (159 C>G (SEQ ID NO:6);T53R (SEQ ID NO:8)) (134 C>G (SEQ ID NO:2); T45R (SEQ ID NO:4)) in twounrelated ADHSP kindreds that disrupts an inter-species conserved aminoacid and which was absent in control subjects (N=105) shows thepathogenic significance of the NIPA T53R (SEQ ID NO: 8)/T45R (SEQ IDNO:4) missense mutation. Discovery of NIPA1 mutations as the cause ofSPG6-linked HSP shows an ability to diagnose HSP and to provide geneticcounseling. The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, it is contemplated thatSPG6 arises from altered signal transduction and/or small moleculetransport through a membrane. NIPA-1 and its ligand are an attractivetarget for therapeutic intervention in SPG6 and other spasticparaplegias. Identification of the NIPA-1 cellular and subcellularlocalization, function and ligand will aid an understanding of axonalneurodegeneration in HSP and will have important therapeuticimplications.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

We claim:
 1. A nucleic acid molecule consisting of at least 30contiguous nucleotides of SEQ ID NO: 2, wherein the nucleic acidincludes position 134 of SEQ ID NO: 2 and wherein said nucleic acid isdetectably labeled with a reporter molecule, wherein the reportermolecule is enzymatic, fluorescent, radioactive, and/or luminescent. 2.The nucleic acid molecule of claim 1, wherein the nucleic acid consistsof at least 50 contiguous nucleotides of SEQ ID NO:
 2. 3. The nucleicacid molecule of claim 1, wherein the nucleic acid molecule isimmobilized to a solid support.
 4. The nucleic acid molecule of claim 3,wherein the solid support is a DNA chip.
 5. A kit for analyzing risk ofneurological disorders comprising at least one nucleic acid molecule ofclaim
 1. 6. The kit of claim 5, further comprising a buffering agents,nucleic acid stabilizing reagents, protein stabilizing reagents, asignal producing reagent, and/or a positive control sample.